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Prostanoid production in umbilical arteries from preterm and term deliveries perfused in vitro
Early Human Development. 24 (1990) 153-161
153
Elsevier Scientific Publishers Ireland Ltd.
EHD01114
Prostanoid production in umbilical arteries from preterm and term deliveries perfused in vitro G. Haugena, S. Stray-Pedersen” and K. Bj@rob Wepartment of Obstetrics and Gynecology and Vnstitute of Clinical Biochemistry, National Hospital, University of Oslo. Oslo (Norway) (Received 20 April 1990; revision received 16 July 1990; accepted 12 September 1990)
Summary
The endogenous prostanoid production in human umbilical arteries from gestational stages between 192 and 290 days was investigated using an in vitro perfusion technique. The outflow concentrations of prostacyclin and thromboxane (measured as their stable metabolites 6-keto-PGF,. and TXB,), PGE, and PGF,. were determined by RIA-methods. The production of all four prostanoids was found to increase with the duration of pregnancy. The formation of prostacyclin was always dominating, followed by PGE,, PGFzo and thromboxane. prematurity; prostanoid production; umbilical artery.
Introduction
At term human umbilical arteries are capable of producing substantial amounts of vasoactive prostanoids [ 1,2]. These endogenous prostanoids may function as local hormones influencing vascular smooth muscle tension and thereby umbilical blood flow. The synthetic capacity of the umbilico-placental vasculature at earlier stages of pregnancy has been little investigated. Studies pertaining to incubation of human umbilical arterial strips [3,4] and superfusion studies of placental tissue [5] have failed to demonstrate any correlation between gestational age and the Correspondence
National Hospital,
to: Guttorm
Haugen, Department 0027 Oslo 1, Norway.
of Obstetrics and Gynecology,
0378-3782/90/$03.50 0 1990 Elsevier Scientific Publishers Published and Printed in Ireland
Ireland Ltd.
University
of Oslo,
154
production of prostacyclin. On the other hand, ultrastructural studies indicate the existence of a higher metabolic activity in endothelial cells from umbilical arteries at term as compared to that observed in vessels obtained from second trimester pregnancies [a]. In this investigation the vascular production of prostacyclin, thromboxane, PGE, and PGF, has been studied during in vitro perfusion of umbilical arteries obtained after preterm deliveries. The results were compared to those observed in cord arteries from term pregnancies. Material and Methods Study groups The investigation was carried out on umbilical cords from three different groups of pregnancies: Group 1: The verypreterm group consisted of four women delivering at 192-230 days’ gestation. All women had received one or more injections with 12 mg of betamethasone before delivery in order to accelerate fetal lung maturity. They were also given tocolytic treatment with fl-sympaticomimetics (fenoterol or terbutalin) in combination with a calcium channel blocker (verapamil) in doses commonly recommended [7,8]. Group 2: The moderately preterm group comprised six women delivering at 237-244 days’ gestation. None of these women had received glucocorticoid or tocolytic treatment. Group 3: The group at term comprised eight women with uncomplicated single pregnancies who delivered at 259-290 days’ gestation. Some relevant clinical data for the groups are listed in Table I. The median age of the women was 30 years (range 20-36 years). Nine of them were nulliparous, seven were primiparous and two were multiparous. They were all normotensive and without proteinuria. Except for the glucocorticoid and tocolytic therapy given to the women in Group 1, they had received no drugs known to interfere with prostanoid synthesis during the last three weeks prior to delivery. Perfusion procedure and prostanoidproduction Umbilical cords were collected immediately after delivery and placed in ice-cold Ringer solution. The segment to be perfused had a length of about 10 cm and was usually cut from the intermediate part of the cord. From the singleton pregnancies two segments were employed, from the twin and triplet pregnancies only one segment was employed from each cord. One of the arteries was cannulated at both ends of the segment. In the preterm groups the cannulation procedure was often technically difficult due to fragility of the arteries. The preparation was then immersed in a phosphate/bicarbonate-buffered electrolyte solution. A serum-free cell culture medium was used as perfusate (Fischer’s medium for leukemic cells of mice). The vessels were perfused at a constant flow of 1 ml/min, giving perfusion pressures below 20 mmHg. The perfusate and the immersion fluid were saturated with a gas
155 TABLE I Clinical data for the three study groups. Patient No.
Reason for admittance
Mode of delivery’
Analgesia/ anaesthesia
C.S. C.S.
Epidural General
C.S.
General
V.D.
General
2880 2280 2740 2320
V.D. C.S. V.D. V.D.
I 1970 II 1940 III 1890 I 1040 II 1510
C.S.
Epidural General Perineal Nitrous oxide Epidural
C.S.
Epidural
C.S.
Epidural (n = 3) Pudendal/ perineal (n = 5)
Oest.age at delivery
Birth weight
(days)
(e)
Group I: Very pretem group (betamethasone treat&) 1615 220 PROM 1 900 192 Placenta 2 praevia IloW 192 Preterm 3 I 1ozob contract. 12010 230 PROM 4 II l!xlo Group 2: Moderately preterm group 237 PROM 1 243 PROM 2 241 PROM 3 242 PROM 4 5
Preterm contract.
243
6
IUGR
241
Group 3: Group at term (median and range values) 279 g= 8 (259-290)
3645 (3400-4290)
(n = 2)
F.S. = rxsaman section. V.D. = vaginal delivery. bNeonatal death on 4th day due to septic infection. ‘Moderate RDS.
mixture of 95% 0, and 5% CO, keeping the pH constant at 7.4. All experiments were carried out at 37 OC. The perfusion was established within 1 h after delivery and the experiments lasted for 8 h. A more detailed description of the perfusion technique has been published earlier [ 11. Measurements of prostanoid production The effluents were collected in polypropylene tubes in automatic fraction collectors in 5-min fractions every 30th min and stored at - 20°C for two weeks at most before analysis. The concentrations of 6-keto-PGF,. and TXB, (the stable metabolites of prostacyclin and thromboxane, respectively), PGE, and PGF, were
156
determined by radio-immunoassay methods [l]. The lower limits of detection were 40 pg/ml for bketo-PGF,. and PGE,, 15 pg/ml for TXB, and 20 pg/ml for PGF,. The inter- and intra-assay coefficients of variation were 10% and 7070,respectively, for 6-keto-PGF,., 11% and 7% for PGE,, 12% and 8% for TXB, and 11% and 6% for PGF,. The concentration values were expressed in pg per ml per cm of arterial length. Measurements of vascular dimensions In a separate study attempts were performed to determine the luminal surface area of both umbilical arteries from 10 cords obtained at 198-290 days’ gestation. The intravascular volume in cord segments with lengths of 25-50 cm was measured as follows: the arteries were cannulated and perfused for 15 min, filled with perfusate under a constant pressure of 70 mmHg, then emptied by injection of air and the fluid content weighed. The inter-assay coefficient of variation of these measurements was 3% (n = 10). The vessel length was determined by catheterization of the entire artery segment. Drugs Prostanoid tracers were purchased from Amersham and prostanoid from Institute of Isotopes, Budapest.
antibodies
Statistical methods Wilcoxon’s rank sum test for unpaired data and linear regression were used for statistical analysis, and P-values below 0.05 were considered as statistically significant.
Prostanoidproduction In all groups the prostanoid concentration levels in the arterial effluents were found to follow a characteristic pattern throughout the experiments, as is illustrated for 6-keto-PGF,. in Fig. 1. In the beginning of the perfusions the concentrations were high, especially those of 6-keto-PGF,11. These high values could most probably be ascribed to an increased release of prostanoids due to inevitable tissue damage during the cannulation and mechanical manipulation of the preparations before the perfusion. Then the concentrations declined and stabilized after 180 min of perfusion. The levels recorded during the stable stage were considered to reflect the basal prostanoid production rate of the umbilical arteries. Table II presents the median prostanoid concentration values for the different groups obtained after 240 min until 360 min of perfusion. As may be observed from the table, all four prostanoids could be detected in the arterial effluents. The prostanoid concentrations were highest at term (Group 3) and lowest at 192-230 days’ gestation (Group 1). In all three groups 6-keto-PGF,o was dominating, whereas the levels of the other prostanoids were encountered in the following sequence: PGE,, PGF,. and TXB,.
157
o-----o o-o m-m
60
120
180
240 300 Time (min.)
360
Group 1 Group 2 Group 3
420
480
Fig. 1. The concentrations throughout
of 6-keto-PGF,a obtained in the outflow samples in the different groups the perfusion experiments (mean values and S.E.W.
TABLE 11 Prostanoid production (pg ml-’ cm-‘) in the umbilical arteries from the three groups (median and range values). R = number of arteries investigated. Group 1 (very preterm) u= 8
Group 2 (moder. preterm) n = 13
Group 3 (term) n = 16
30.8 (8.3-73.0)
50.6 (32.8-74.8)
159.5 (117.0-240.0) P<
NS
PGE,
11.5 (6.8-16.3)
0.01 38.1 (20.6-54.2)
32.9 (17.1-58.0) P<
NS
0.01
17.1 (9.8-26.2)
PGF, (;::-6.7)
(Z22.8) P<
TXB,
P<
0.01
P<
0.01
3.1 (1.8-7.8) P<
6-kcto-PGF,,/TXBi ratio
0.05
0.9 (0.14.2)
0.05
(Z-7.9)
16.9 (5.4-24.7)
35.1 (17.4-124.0) P<
0.01
29.4 (20.5-41.1) P<
0.01
158 TABLLE 111 The prostanoid production (pg ml-’ cm-‘) in the different cords from Group 1. Patient No.
Betarnethasone admin. 12 mg 6 days before delivery 12 mg 3 days before delivery 12 mg 12 hours before delivery 12mg X 3,1ast admin. 5 days before delivery
6-keto-
TXB,
PGE,
PGE,
1.9 1.1 4.2 3.7 0.6 0.1 0.1 0.1
3.9 2.4 4.1 6.7 4.4 3.4 2.2 4.3
10.7 16.3 13.4 11.3 16.1 11.6 7.6 6.8
PGFh Art. Art. Art. Art. Twin Twin Twin Twin
I II I II
1 2 1 2
51.2 42.8 73.0 71.3 18.7 12.4 12.1 8.3
Table III demonstrates the data obtained for each of the four patients in Group 1. As shown, the umbilical artery outflow concentrations of 6-keto-PGF,. and TXB, were markedly lower in the two pregnancies where betamethasone had been administered repeatedly (patient No. 4) or less than 12 h before the delivery (patient No. 3). The values recorded for PGE, and PGF,, displayed less variations. Measurements of luminal surface area Up to 246 days’ gestation the luminal surface area of the umbilical arteries was found to increase linearly with gestational age (r = 0.96, P < 0.001). The median values obtained at gestational ages of I%-220 days (n = 6 arteries), 237-246 days (n = 6) and 279-290 days (n = 8) were 0.94 (range 0.78-l. 19), 1.43 (range 1.391.47) and 1.37 (range 1.19-l .46) cm* per cm of arterial length, respectively. Discussion The present investigation confirmed that human umbilical arteries are capable of prostanoid synthesis from 27 weeks of pregnancy. In all preparations the production of prostacyclin was found to be most pronounced, followed by PGE,, PGFzO and finally by thromboxane. On the other hand, the three groups showed marked differences with respect to the amount as well as to the pattern of the prostanoid production. This contrasts with earlier observations in slices of umbilical arteries [3,4]. Admittedly, the prostanoids measured in these earlier studies might have originated from different parts of the vessel wall, e.g. the smooth muscle cells. When employing the in vitro perfusion technique, it seems reasonable to assume that the prostanoids released into the effluents predominantly reflected the production rate of the endothelial cells. The observed variations in luminal surface area might be of importance when comparing the production rates of the three study groups. In accord with earlier ultrasound
159
measurements of the human umbilical vein diameter [9], the vascular dimensions were found to increase linearly from the 28th to the 34th week of pregnancy. At term the values were more or less unchanged from the dimensions obtained at 237-246 days’ gestation. However, the same significant differences were maintained when the prostanoid production levels were related to the surface area estimated for the individual groups. The results obtained in the moderately preterm group (Group 2) and the group at term (Group 3) may be compared directly since none of these patients received drugs known to interfere with prostanoid synthesis. The high umbilical arterial PGE,-production obtained in both groups represents an interesting finding, since circulating PGE, is considered to be of importance for the preservation of a patent ductus arteriosus [lo]. Group 2 demonstrated a significantly lower prostacyclin/thromboxaneratio which is considered to be associated with increased platelet aggregation and vascular smooth muscle tonus. This ratio, however, has to be interpreted with some caution, since we did not measure the synthetic activity of the platelets which are known to be the principal source of thromboxane. The prostanoid production pattern obtained in the very preterm group (Group 1) deviated considerably from that observed in the moderately preterm group (Group 2). In the former group lower prostanoid levels were encountered, except for PGF,, whereas the prostacyclin/thromboxane ratio was found to be significantly higher. All of the four women, however, had received treatment which could have influenced prostanoid metabolism, such as P-sympaticomimetics, calcium channel blockers and glucocorticoids. These drugs pass the placental barrier rapidly [7,11]. A more detailed discussion about their possible influence on fetal vascular prostanoid production seems therefore justified. As for the tocolytic drugs, no changes in the fetal plasma levels of 6-keto-PGF,. or TXB, were observed following short term ritodrine infusion in a study among women undergoing elective caesarean section [12]. It has also been demonstrated that fl-sympaticomimetics in doses up to 10S4M do not affect thromboxane generation in fetal platelets or prostacyclin synthesis in superfused slices of umbilical arteries [ 131. Drugs affecting cellular calcium metabolism might interfere with prostanoid synthesis since calcium ions activate phospholipases. This has been confirmed by the detection of an increased prostacyclin production in cultured endothelial cells after addition of a calcium ionophore [14]. On the other hand, the prostacyclin production in incubated rings of umbilical veins has been found to be stimulated by diltiazem, a calcium channel blocker [15]. In our study the fetal blood concentrations of calcium channel blockers must have been very low [l l] as judged from the doses given to their mothers and also from the time interval between the last administration and the delivery. Thus, neither calcium channel blockers nor /3-sympaticomimetics seemed to be of any major importance for the umbilical arterial prostanoid production in Group 1. Dexamethasone and hydrocortisone prevent the release of arachidonic acid by inhibiting phospholipase activity. In vitro experiments with addition of either of the drugs to superfused strips of umbilical arteries have revealed a depression of prostacyclin synthesis, albeit the doses employed were higher than those employed thera-
160
peutically [ 131. The present study might suggest that glucocorticoids have an impact on prostanoid production in umbilical arteries since the outflow prostanoid levels were found to be correlated to the betamethasone treatment, both with respect to the number of doses given, as well as to the time duration from the last injection to delivery. The increase in the prostacyclinkhromboxane-ratio observed in Group 1 might thus perhaps be considered as an additional beneficial effect of glucocorticoid treatment on the fetal outcome in extremely preterm pregnancies. However, these results have to be interpreted with caution due to the small number of data. In conclusion, the present investigation demonstrates that the prostanoid synthetic capacity of umbilical endothelial cells is dependent on gestational age. In fullterm pregnancies both prostacyclin and thromboxane affect umbilico-placental circulation [2] and endogenously produced prostanoids have been shown to be involved in pressure responses to several autacoids in these vessels [la]. The different production pattern in preterm umbilical arteries might therefore cause alterations in the responsiveness to vasoactive substances, as has in fact recently been reported [ 171. Acknowledgments The study was supported by The Norwegian Council on Cardiovascular Diseases, Anders Jahre’s Foundation, the Norwegian Research Council for Science and the Humanities and The Society for the Protection of Animals. References Bjfiro, K., Hovig, T., Stokke. K.T. and Stray-Pedersen, S. (1986): Formation of prostanoids in human umbilical vessels perfused in vitro. Prostaglandins, 3 1.683-698. Tuvemo. T. (1980): Role of prostaglandins, prostacychn. and thromboxanes in the control of the umbilical-placental circulation. Semin. Perinatol., 4,91-95. Ongari, M.A.. Ritter, J.M., Orchard, M.A., Waddell, K.A., Blair, LA. and Lewis, P.J. (1984): Correlation of prostacyclin synthesis by human umbilical artery with status of essential fatty acid. Am. J. Obstet. Gynecol.. 149,455-460. Stuart. M.J., Sunderji, S.G., Yambo, T., Clark, D.A., Allen, J.B., Elrad, H. and Slott, J.H. (1981): Decreased prostacyclin production: A characteristic of chronic placental insufficiency syndromes. Lancet, i, 11261128. M%kill, U.-M. Jouppila, P., Kirkinen, P., Viinikka, L. and Ylikorkala, 0. (1986): Placenta) thromboxane and prostacyclin in the regulation of placenta) blood flow. Obstet. Gynecol., 68, 537 - 540. Parry, E.W. and Abramovich, D.R. (1972): The ultrastructure of human umbilical vessel endothehum from early pregnancy to full term. J. Anat., 111,29-42. Caritis, S.N. (1983): Treatment of preterm labour. A review of the therapeutic options. Drugs, 26, 243-261. Gummerus, M. (1977): Die Behandlung der vorxeitigen Wehentatigkeit und Antagonisierung der Nebenwirkungen der tokolytischen Therapie mit Verapamil. Z. Geburtshilfe Perinatol., 181, 334340. Gill, R.W., Trudinger, B.J., Garrett, W.J., Kossoff, G. and Warren, P.S. (1981): Fetal umbilical venous flow measured in utero by pulsed Doppler and B-mode ultrasound. I. Normal pregnancies. Am. J. Obstet. Gynecol., 139,720-725.
161 Clyman, R.I. (1987): Ductus arteriosus: Current theories of prenatal and postnatal regulation. Semin. Perinatol., 11,64-71. 11 Strigl. R.. Gastroph, G., Hege. H.G., Doring, P. and Mehring, W. (1980): Nachweis von Verapamil in mutterlichen und fetalen Blut des Menschen. Geburtshilfe Frauenheikd., 40,496-499. 12 Ekblad, U., Erkkola, R., Uotila. P., Kanto, J. and Palo, P. (1988): Ritodrine infusion at term: Effects on maternal and fetal prostacyclin, thromboxane and prostaglandin precursor fatty acids. Gynecol. Obstet. Invest., 25, 106-l 12. 13 M&i&i. U.-M. (1984): The effects of betamimetics and glucocorticoids on fetal vascular prostacyclin and platelet thromboxane synthesis in humans. Prostaglandins Leukotrienes Med., 16, 1l-17. 14 Weksler, B.B., Ley, C.W. and Jaffe, E.A. (1978): Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A 23187. J. Clin. Invest., 62,923-930. 15 Mehta, J., Mehta, P. and Ostrowski. N. (1986): Calcium blocker diltiazem inhibits platelet activation and stimulates vascular prostacyclin synthesis. Am. J. Med. Sci., 291,20-24. 16 Bjdro, K. and Stray-Pedersen, S. (1986): Characterixation of the responses to serotonin and prostanoids in human umbilical arteries perfused in vitro. Stand. J. Clin. Lab. Invest., 46,85-90. 17 White, R.P. (1989): Pharmacodynamic study of maturation and closure of human umbilical arteries. Am. J. Obstet. Gynecol., 160,229-237.
10
153
Elsevier Scientific Publishers Ireland Ltd.
EHD01114
Prostanoid production in umbilical arteries from preterm and term deliveries perfused in vitro G. Haugena, S. Stray-Pedersen” and K. Bj@rob Wepartment of Obstetrics and Gynecology and Vnstitute of Clinical Biochemistry, National Hospital, University of Oslo. Oslo (Norway) (Received 20 April 1990; revision received 16 July 1990; accepted 12 September 1990)
Summary
The endogenous prostanoid production in human umbilical arteries from gestational stages between 192 and 290 days was investigated using an in vitro perfusion technique. The outflow concentrations of prostacyclin and thromboxane (measured as their stable metabolites 6-keto-PGF,. and TXB,), PGE, and PGF,. were determined by RIA-methods. The production of all four prostanoids was found to increase with the duration of pregnancy. The formation of prostacyclin was always dominating, followed by PGE,, PGFzo and thromboxane. prematurity; prostanoid production; umbilical artery.
Introduction
At term human umbilical arteries are capable of producing substantial amounts of vasoactive prostanoids [ 1,2]. These endogenous prostanoids may function as local hormones influencing vascular smooth muscle tension and thereby umbilical blood flow. The synthetic capacity of the umbilico-placental vasculature at earlier stages of pregnancy has been little investigated. Studies pertaining to incubation of human umbilical arterial strips [3,4] and superfusion studies of placental tissue [5] have failed to demonstrate any correlation between gestational age and the Correspondence
National Hospital,
to: Guttorm
Haugen, Department 0027 Oslo 1, Norway.
of Obstetrics and Gynecology,
0378-3782/90/$03.50 0 1990 Elsevier Scientific Publishers Published and Printed in Ireland
Ireland Ltd.
University
of Oslo,
154
production of prostacyclin. On the other hand, ultrastructural studies indicate the existence of a higher metabolic activity in endothelial cells from umbilical arteries at term as compared to that observed in vessels obtained from second trimester pregnancies [a]. In this investigation the vascular production of prostacyclin, thromboxane, PGE, and PGF, has been studied during in vitro perfusion of umbilical arteries obtained after preterm deliveries. The results were compared to those observed in cord arteries from term pregnancies. Material and Methods Study groups The investigation was carried out on umbilical cords from three different groups of pregnancies: Group 1: The verypreterm group consisted of four women delivering at 192-230 days’ gestation. All women had received one or more injections with 12 mg of betamethasone before delivery in order to accelerate fetal lung maturity. They were also given tocolytic treatment with fl-sympaticomimetics (fenoterol or terbutalin) in combination with a calcium channel blocker (verapamil) in doses commonly recommended [7,8]. Group 2: The moderately preterm group comprised six women delivering at 237-244 days’ gestation. None of these women had received glucocorticoid or tocolytic treatment. Group 3: The group at term comprised eight women with uncomplicated single pregnancies who delivered at 259-290 days’ gestation. Some relevant clinical data for the groups are listed in Table I. The median age of the women was 30 years (range 20-36 years). Nine of them were nulliparous, seven were primiparous and two were multiparous. They were all normotensive and without proteinuria. Except for the glucocorticoid and tocolytic therapy given to the women in Group 1, they had received no drugs known to interfere with prostanoid synthesis during the last three weeks prior to delivery. Perfusion procedure and prostanoidproduction Umbilical cords were collected immediately after delivery and placed in ice-cold Ringer solution. The segment to be perfused had a length of about 10 cm and was usually cut from the intermediate part of the cord. From the singleton pregnancies two segments were employed, from the twin and triplet pregnancies only one segment was employed from each cord. One of the arteries was cannulated at both ends of the segment. In the preterm groups the cannulation procedure was often technically difficult due to fragility of the arteries. The preparation was then immersed in a phosphate/bicarbonate-buffered electrolyte solution. A serum-free cell culture medium was used as perfusate (Fischer’s medium for leukemic cells of mice). The vessels were perfused at a constant flow of 1 ml/min, giving perfusion pressures below 20 mmHg. The perfusate and the immersion fluid were saturated with a gas
155 TABLE I Clinical data for the three study groups. Patient No.
Reason for admittance
Mode of delivery’
Analgesia/ anaesthesia
C.S. C.S.
Epidural General
C.S.
General
V.D.
General
2880 2280 2740 2320
V.D. C.S. V.D. V.D.
I 1970 II 1940 III 1890 I 1040 II 1510
C.S.
Epidural General Perineal Nitrous oxide Epidural
C.S.
Epidural
C.S.
Epidural (n = 3) Pudendal/ perineal (n = 5)
Oest.age at delivery
Birth weight
(days)
(e)
Group I: Very pretem group (betamethasone treat&) 1615 220 PROM 1 900 192 Placenta 2 praevia IloW 192 Preterm 3 I 1ozob contract. 12010 230 PROM 4 II l!xlo Group 2: Moderately preterm group 237 PROM 1 243 PROM 2 241 PROM 3 242 PROM 4 5
Preterm contract.
243
6
IUGR
241
Group 3: Group at term (median and range values) 279 g= 8 (259-290)
3645 (3400-4290)
(n = 2)
F.S. = rxsaman section. V.D. = vaginal delivery. bNeonatal death on 4th day due to septic infection. ‘Moderate RDS.
mixture of 95% 0, and 5% CO, keeping the pH constant at 7.4. All experiments were carried out at 37 OC. The perfusion was established within 1 h after delivery and the experiments lasted for 8 h. A more detailed description of the perfusion technique has been published earlier [ 11. Measurements of prostanoid production The effluents were collected in polypropylene tubes in automatic fraction collectors in 5-min fractions every 30th min and stored at - 20°C for two weeks at most before analysis. The concentrations of 6-keto-PGF,. and TXB, (the stable metabolites of prostacyclin and thromboxane, respectively), PGE, and PGF, were
156
determined by radio-immunoassay methods [l]. The lower limits of detection were 40 pg/ml for bketo-PGF,. and PGE,, 15 pg/ml for TXB, and 20 pg/ml for PGF,. The inter- and intra-assay coefficients of variation were 10% and 7070,respectively, for 6-keto-PGF,., 11% and 7% for PGE,, 12% and 8% for TXB, and 11% and 6% for PGF,. The concentration values were expressed in pg per ml per cm of arterial length. Measurements of vascular dimensions In a separate study attempts were performed to determine the luminal surface area of both umbilical arteries from 10 cords obtained at 198-290 days’ gestation. The intravascular volume in cord segments with lengths of 25-50 cm was measured as follows: the arteries were cannulated and perfused for 15 min, filled with perfusate under a constant pressure of 70 mmHg, then emptied by injection of air and the fluid content weighed. The inter-assay coefficient of variation of these measurements was 3% (n = 10). The vessel length was determined by catheterization of the entire artery segment. Drugs Prostanoid tracers were purchased from Amersham and prostanoid from Institute of Isotopes, Budapest.
antibodies
Statistical methods Wilcoxon’s rank sum test for unpaired data and linear regression were used for statistical analysis, and P-values below 0.05 were considered as statistically significant.
Prostanoidproduction In all groups the prostanoid concentration levels in the arterial effluents were found to follow a characteristic pattern throughout the experiments, as is illustrated for 6-keto-PGF,. in Fig. 1. In the beginning of the perfusions the concentrations were high, especially those of 6-keto-PGF,11. These high values could most probably be ascribed to an increased release of prostanoids due to inevitable tissue damage during the cannulation and mechanical manipulation of the preparations before the perfusion. Then the concentrations declined and stabilized after 180 min of perfusion. The levels recorded during the stable stage were considered to reflect the basal prostanoid production rate of the umbilical arteries. Table II presents the median prostanoid concentration values for the different groups obtained after 240 min until 360 min of perfusion. As may be observed from the table, all four prostanoids could be detected in the arterial effluents. The prostanoid concentrations were highest at term (Group 3) and lowest at 192-230 days’ gestation (Group 1). In all three groups 6-keto-PGF,o was dominating, whereas the levels of the other prostanoids were encountered in the following sequence: PGE,, PGF,. and TXB,.
157
o-----o o-o m-m
60
120
180
240 300 Time (min.)
360
Group 1 Group 2 Group 3
420
480
Fig. 1. The concentrations throughout
of 6-keto-PGF,a obtained in the outflow samples in the different groups the perfusion experiments (mean values and S.E.W.
TABLE 11 Prostanoid production (pg ml-’ cm-‘) in the umbilical arteries from the three groups (median and range values). R = number of arteries investigated. Group 1 (very preterm) u= 8
Group 2 (moder. preterm) n = 13
Group 3 (term) n = 16
30.8 (8.3-73.0)
50.6 (32.8-74.8)
159.5 (117.0-240.0) P<
NS
PGE,
11.5 (6.8-16.3)
0.01 38.1 (20.6-54.2)
32.9 (17.1-58.0) P<
NS
0.01
17.1 (9.8-26.2)
PGF, (;::-6.7)
(Z22.8) P<
TXB,
P<
0.01
P<
0.01
3.1 (1.8-7.8) P<
6-kcto-PGF,,/TXBi ratio
0.05
0.9 (0.14.2)
0.05
(Z-7.9)
16.9 (5.4-24.7)
35.1 (17.4-124.0) P<
0.01
29.4 (20.5-41.1) P<
0.01
158 TABLLE 111 The prostanoid production (pg ml-’ cm-‘) in the different cords from Group 1. Patient No.
Betarnethasone admin. 12 mg 6 days before delivery 12 mg 3 days before delivery 12 mg 12 hours before delivery 12mg X 3,1ast admin. 5 days before delivery
6-keto-
TXB,
PGE,
PGE,
1.9 1.1 4.2 3.7 0.6 0.1 0.1 0.1
3.9 2.4 4.1 6.7 4.4 3.4 2.2 4.3
10.7 16.3 13.4 11.3 16.1 11.6 7.6 6.8
PGFh Art. Art. Art. Art. Twin Twin Twin Twin
I II I II
1 2 1 2
51.2 42.8 73.0 71.3 18.7 12.4 12.1 8.3
Table III demonstrates the data obtained for each of the four patients in Group 1. As shown, the umbilical artery outflow concentrations of 6-keto-PGF,. and TXB, were markedly lower in the two pregnancies where betamethasone had been administered repeatedly (patient No. 4) or less than 12 h before the delivery (patient No. 3). The values recorded for PGE, and PGF,, displayed less variations. Measurements of luminal surface area Up to 246 days’ gestation the luminal surface area of the umbilical arteries was found to increase linearly with gestational age (r = 0.96, P < 0.001). The median values obtained at gestational ages of I%-220 days (n = 6 arteries), 237-246 days (n = 6) and 279-290 days (n = 8) were 0.94 (range 0.78-l. 19), 1.43 (range 1.391.47) and 1.37 (range 1.19-l .46) cm* per cm of arterial length, respectively. Discussion The present investigation confirmed that human umbilical arteries are capable of prostanoid synthesis from 27 weeks of pregnancy. In all preparations the production of prostacyclin was found to be most pronounced, followed by PGE,, PGFzO and finally by thromboxane. On the other hand, the three groups showed marked differences with respect to the amount as well as to the pattern of the prostanoid production. This contrasts with earlier observations in slices of umbilical arteries [3,4]. Admittedly, the prostanoids measured in these earlier studies might have originated from different parts of the vessel wall, e.g. the smooth muscle cells. When employing the in vitro perfusion technique, it seems reasonable to assume that the prostanoids released into the effluents predominantly reflected the production rate of the endothelial cells. The observed variations in luminal surface area might be of importance when comparing the production rates of the three study groups. In accord with earlier ultrasound
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measurements of the human umbilical vein diameter [9], the vascular dimensions were found to increase linearly from the 28th to the 34th week of pregnancy. At term the values were more or less unchanged from the dimensions obtained at 237-246 days’ gestation. However, the same significant differences were maintained when the prostanoid production levels were related to the surface area estimated for the individual groups. The results obtained in the moderately preterm group (Group 2) and the group at term (Group 3) may be compared directly since none of these patients received drugs known to interfere with prostanoid synthesis. The high umbilical arterial PGE,-production obtained in both groups represents an interesting finding, since circulating PGE, is considered to be of importance for the preservation of a patent ductus arteriosus [lo]. Group 2 demonstrated a significantly lower prostacyclin/thromboxaneratio which is considered to be associated with increased platelet aggregation and vascular smooth muscle tonus. This ratio, however, has to be interpreted with some caution, since we did not measure the synthetic activity of the platelets which are known to be the principal source of thromboxane. The prostanoid production pattern obtained in the very preterm group (Group 1) deviated considerably from that observed in the moderately preterm group (Group 2). In the former group lower prostanoid levels were encountered, except for PGF,, whereas the prostacyclin/thromboxane ratio was found to be significantly higher. All of the four women, however, had received treatment which could have influenced prostanoid metabolism, such as P-sympaticomimetics, calcium channel blockers and glucocorticoids. These drugs pass the placental barrier rapidly [7,11]. A more detailed discussion about their possible influence on fetal vascular prostanoid production seems therefore justified. As for the tocolytic drugs, no changes in the fetal plasma levels of 6-keto-PGF,. or TXB, were observed following short term ritodrine infusion in a study among women undergoing elective caesarean section [12]. It has also been demonstrated that fl-sympaticomimetics in doses up to 10S4M do not affect thromboxane generation in fetal platelets or prostacyclin synthesis in superfused slices of umbilical arteries [ 131. Drugs affecting cellular calcium metabolism might interfere with prostanoid synthesis since calcium ions activate phospholipases. This has been confirmed by the detection of an increased prostacyclin production in cultured endothelial cells after addition of a calcium ionophore [14]. On the other hand, the prostacyclin production in incubated rings of umbilical veins has been found to be stimulated by diltiazem, a calcium channel blocker [15]. In our study the fetal blood concentrations of calcium channel blockers must have been very low [l l] as judged from the doses given to their mothers and also from the time interval between the last administration and the delivery. Thus, neither calcium channel blockers nor /3-sympaticomimetics seemed to be of any major importance for the umbilical arterial prostanoid production in Group 1. Dexamethasone and hydrocortisone prevent the release of arachidonic acid by inhibiting phospholipase activity. In vitro experiments with addition of either of the drugs to superfused strips of umbilical arteries have revealed a depression of prostacyclin synthesis, albeit the doses employed were higher than those employed thera-
160
peutically [ 131. The present study might suggest that glucocorticoids have an impact on prostanoid production in umbilical arteries since the outflow prostanoid levels were found to be correlated to the betamethasone treatment, both with respect to the number of doses given, as well as to the time duration from the last injection to delivery. The increase in the prostacyclinkhromboxane-ratio observed in Group 1 might thus perhaps be considered as an additional beneficial effect of glucocorticoid treatment on the fetal outcome in extremely preterm pregnancies. However, these results have to be interpreted with caution due to the small number of data. In conclusion, the present investigation demonstrates that the prostanoid synthetic capacity of umbilical endothelial cells is dependent on gestational age. In fullterm pregnancies both prostacyclin and thromboxane affect umbilico-placental circulation [2] and endogenously produced prostanoids have been shown to be involved in pressure responses to several autacoids in these vessels [la]. The different production pattern in preterm umbilical arteries might therefore cause alterations in the responsiveness to vasoactive substances, as has in fact recently been reported [ 171. Acknowledgments The study was supported by The Norwegian Council on Cardiovascular Diseases, Anders Jahre’s Foundation, the Norwegian Research Council for Science and the Humanities and The Society for the Protection of Animals. References Bjfiro, K., Hovig, T., Stokke. K.T. and Stray-Pedersen, S. (1986): Formation of prostanoids in human umbilical vessels perfused in vitro. Prostaglandins, 3 1.683-698. Tuvemo. T. (1980): Role of prostaglandins, prostacychn. and thromboxanes in the control of the umbilical-placental circulation. Semin. Perinatol., 4,91-95. Ongari, M.A.. Ritter, J.M., Orchard, M.A., Waddell, K.A., Blair, LA. and Lewis, P.J. (1984): Correlation of prostacyclin synthesis by human umbilical artery with status of essential fatty acid. Am. J. Obstet. Gynecol.. 149,455-460. Stuart. M.J., Sunderji, S.G., Yambo, T., Clark, D.A., Allen, J.B., Elrad, H. and Slott, J.H. (1981): Decreased prostacyclin production: A characteristic of chronic placental insufficiency syndromes. Lancet, i, 11261128. M%kill, U.-M. Jouppila, P., Kirkinen, P., Viinikka, L. and Ylikorkala, 0. (1986): Placenta) thromboxane and prostacyclin in the regulation of placenta) blood flow. Obstet. Gynecol., 68, 537 - 540. Parry, E.W. and Abramovich, D.R. (1972): The ultrastructure of human umbilical vessel endothehum from early pregnancy to full term. J. Anat., 111,29-42. Caritis, S.N. (1983): Treatment of preterm labour. A review of the therapeutic options. Drugs, 26, 243-261. Gummerus, M. (1977): Die Behandlung der vorxeitigen Wehentatigkeit und Antagonisierung der Nebenwirkungen der tokolytischen Therapie mit Verapamil. Z. Geburtshilfe Perinatol., 181, 334340. Gill, R.W., Trudinger, B.J., Garrett, W.J., Kossoff, G. and Warren, P.S. (1981): Fetal umbilical venous flow measured in utero by pulsed Doppler and B-mode ultrasound. I. Normal pregnancies. Am. J. Obstet. Gynecol., 139,720-725.
161 Clyman, R.I. (1987): Ductus arteriosus: Current theories of prenatal and postnatal regulation. Semin. Perinatol., 11,64-71. 11 Strigl. R.. Gastroph, G., Hege. H.G., Doring, P. and Mehring, W. (1980): Nachweis von Verapamil in mutterlichen und fetalen Blut des Menschen. Geburtshilfe Frauenheikd., 40,496-499. 12 Ekblad, U., Erkkola, R., Uotila. P., Kanto, J. and Palo, P. (1988): Ritodrine infusion at term: Effects on maternal and fetal prostacyclin, thromboxane and prostaglandin precursor fatty acids. Gynecol. Obstet. Invest., 25, 106-l 12. 13 M&i&i. U.-M. (1984): The effects of betamimetics and glucocorticoids on fetal vascular prostacyclin and platelet thromboxane synthesis in humans. Prostaglandins Leukotrienes Med., 16, 1l-17. 14 Weksler, B.B., Ley, C.W. and Jaffe, E.A. (1978): Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A 23187. J. Clin. Invest., 62,923-930. 15 Mehta, J., Mehta, P. and Ostrowski. N. (1986): Calcium blocker diltiazem inhibits platelet activation and stimulates vascular prostacyclin synthesis. Am. J. Med. Sci., 291,20-24. 16 Bjdro, K. and Stray-Pedersen, S. (1986): Characterixation of the responses to serotonin and prostanoids in human umbilical arteries perfused in vitro. Stand. J. Clin. Lab. Invest., 46,85-90. 17 White, R.P. (1989): Pharmacodynamic study of maturation and closure of human umbilical arteries. Am. J. Obstet. Gynecol., 160,229-237.
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