Fetal prostaglandin levels in twin pregnancies

Prostaglandins and Medicine 6:

FETAL

309-316, 1981

PROSTAGLANDIN LEVELS

IN

TWIN

PREGNANCIES

R.J. Norman, B.L.F. Bredenkamp, S.M. Joubert, C. Beetar. Department of Chemical Pathology, University of Natal, P 0 Box 17039, Congella, 4013, Republic of South Africa. (reprint requests to RJN). ABSTRACT Umbilical venous plasma and amniotic fluid concentrations of prostaglandins E2 (PGE), F,, (PGF) and 13,14 dihydro-15-keto-F2, (PGFM) were measured in 31 twin pregnancies by specific radioimnunoassays. There was no significant difference between twins I and II in the same pregnancy but PGE and PGFM were significantly increased during labour in fetal blood. This rise was not manifest in the latent phase. PG concentration in amniotic fluid rose with advancing gestational age but was more pronounced in the case of PGE than for PGF and PGFM. No significant differences between amniotic sac concentrations of PGs of twin I and II were found during the prelabour period, but in all four patients in labour with intact membranes, the PG levels were increased in twin I. The fetal plasma and amniotic fluid levels of PGE and PGFM were significantly correlated in the prelabour group. INTRODUCTION Recent studies have suggested that the naturally occurring prostaglan dins may significantly influence fetal development and adaptation to neonatal life (1,2). This may occur by effects on maternal uteroplacental blood flow (3), by regulation of organ development through enzyme induction (4), or by direct action to alter fetal and placenta1 endocrine function (1). Much of this work has been performed in nonhuman species, particularly the fetal lamb. Investigation of the physiological influence of arachidonic acid . . derivatives has been made possible by the development of sensitive and specific radioimnunoassays for primary prostaglandins and their metabolites (5,6). Several authors have measured circulating levels of prostaglandins by these methods in amniotic fluid (7) and fetal and neonatal blood (8,9). As prostaglandins E, Fpcrand 13,14 dihydro-ketoFPa have all been shown to be elevated in cord blood of neonates born after labour as compared to infants born by elective Caesarean section (lo), this study was designed to compare'prostaglandin values in umbilical 'venousblood and amniotic fluid of twins before and during

309

labour. Determination of prostaglandin levels in multiple pregnancy may be important to shed light on the initiation of labour in humans as well as the physiological function of these compounds in the perinatal period. MATERIALS AND

METHODS

Patients All patients gave consent to this study which was approved by the University ethics committee. Participants were seen at the twin clinic over the last trimester of pregnancy and had uneventful antenatal progress except for eight with mild hypertension. Elective Caesarean section was performed between thirty five and forty weeks for standard obstetric indications including repeat Caesarean section, malpresentations, reduced pelvic dimensions and potential intrauterine growth retardation. Emergency Caesarean section in the active phase of labour was undertaken for cephalo-pelvic disproportion, malpresentations and failure to progress in labour. Anaesthesia was induced with thiopentone and suxamenthonium and maintained with nitrous oxide and oxygen until the babies were born. A wedge was inserted under the right buttock to ensure a 200 lateral tilt. Ten millilitres of umbilical venous blood were drawn into a syringe before clamping of the cord and put on ice in a glass tube containing EDTA,15 mg,and acetylsalicylic acid,0.5 mg. Plasma was immediately separated b centrifugation at 2 5009 for ten minutes at 4oC, frozen at minus 20X C and assayed for prostaglandins within two weeks. About 10 ml of amniotic fluid were obtained by direct amniocentesis and treated in the same manner. All neonates cried at birth; the five minute Apgar score was greater than seven in all babies. Gestational ages were calculated from the last menstrual period and the Dubowitz score. Babies were designated I or II on their order of delivery. Radioimmunoassay of prostaglandins Prostaglandins E2 (PGE),F2a (PGF) and 13,14 dihydro-15-keto-FZa (PGFM) were kindly donated by Dr J Pike, Upjohn Co., Kalamazoo, USA, and were checked for purity by thin layer chromatography. Five milligrams of each were conjugated to bovine serum albumin with water soluble carbodiimide (5) and antibodies raised by multiple intradermal injection into rabbits. Plasma samples (4 ml) at pH 4 were extracted into cyclohexane-ethyl acetate (1 : 1 mixture) and subjected to chromatography as described by Mitchell et al. (6). PGE, PGF and PGFM were eluted as a single fraction. The assay was performed by the method of Dray and colleagu;s5i;). Final antibody titres used were: PGE 1 : 1 500; PGF 1 ; PGFM 1 : 10 000. Tritiated ligands were used throughout (Amersham, specific activity E & F 5.92 TBq/mnol, FM 2.96 TBq/ lmlol). Separation was achieved with dextran coated charcoal and the

310

decanted supernatant counted in a Packard liquid scintillation spectrometer. All glassware was siliconized and polypropylene tubes were used in the incubation procedure. Recoveries were monitored with tritiated PGFM and were always greater than 70%. The ability of a variety of prostaglandins and metabolites to displace the labelled prostaglandin was tested and the relative cross reactivities were calculated from the mass required to displace 50% of the bound label (Table I). The specificity of the determinations reported depends upon the specific characteristics of the antibodies and the use of a chromatographic procedure to separate PGE, PGF and PGFM from interfering substances. The mean of the least detectable mass resulting in a response of two standard deviations from the zero concentration was 2.5 pg for PGF, 5 pg for PGFM and 10 pg for PGE. Estimation of the blank value was less than these and was not subtracted. Intra- and inter-assay coefficients of variation were PGE 10% and 15%, PGF 12% and 18%, PGFM 12% and 20%, respectively. Table I.

Antibody crossreactions to a variety of prostaglandins Anti PGE

El E2

Fla Fsa A2

82

15- keto-F2

13,14 dihy&o E2 13,14 dihydro-15-keto E2 13,14 dihydro-15-keto F2

-4% 100% 4%

1.5% 18%

4%

cl% cl% cl% cl%

Anti PGF

4.1%

Anti PGFM


5%
Statistical analysis The nonparametric tests of Wilcoxon and Mann Whitney were applied to the data and a significance level of 0.05 chosen. RESULTS Blood Umbilical venous levels of PGE, PGF and PGFM at the various stages of labour are recorded in Table II. Although individual values between twins I and II in each group were frequently different from each other, these did not achieve statisticalsignificance (p > 0.05). PGE was present in higher concentrations than PGF but less than PGFM. There was no increase in any of the three prostaglandins in the latent phase of labour (cervix less than 3 cm and not fully effaced) but PGE and PGFM were significantly elevated in active labour when compared to the elective Caesarean section group (p < 0.05 and p c 0.001 respectively).

311

Umbilical venous levels of prostaslandins pregnancies (pg/ml) f SEM .

Table II.

Prelabour

PGE

PGF

(A)

Latent phase

Active phase

(C)

Twin I

Twin

Twin

Twin

Twin

Twin

II

I

II

I

II

139.9

150.0

166.2

150.9

255.4

192.4

k18.2

k22.8

+49.3

+37.3

558.1”

+34.3

Mean = 144.9

Mean = 158.6

f14.4

f29.8

Mean = 222.2 f32.8"

85.6

88.3

108.0

127.5

108.7

95.0

f9.9

f9.4

f25.1

+39.0

f18.6

+8.7

Mean = 87.0

Efean = 117.8

+6.9

PGFM

(6)

in twin

Mean = 101.5

zt21.4

H.8

204.4

216.0

254.1

208.3

497.5

31.6

f22.3

f72.8

k68.0

k121.6""

Mean = 210.2

Mean = 231.2

+22.0

f53.0

455.1 +88.1**

Mean = 475.2 k71.4""

No.in group

(16)

* p ** p SEM 1 PGE = PGFM =

(16)

(5)

(5)

(9)

(10)

< 0.05 compared to corresponding group A < 0.001 compared to corresponding group A Standard er ror of mean Prostagland in E2, PGF = Prostaglandin Fzcr Prostagland in 13,14 dihydro-15-keto F,,

This was reflected ratio (Table III).

in the increased PGE : PGF ratio and PGF : PGFM

Nine of thirty two babies in the prelabour group were less than the 10th centile for gestational age and eight of thirty one pregnancies were complicated by mild hypertension. Prostaglandins in these complicated pregnancies were not significantly different from normal twin pregnancies.

312

Table III.

Prostaglandin ratios in twin pregnancies. (Twins I and II combined). Mean plus standard error of mean Prelabour (A)

Latent phase (B)

Active phase (C)

E:F

1.66 fl.15

1.35 Ho.28

2.52 M.20*

E : FM

0.68 kO.05

0.69 M.16

0.47 kO.O6**

F : FM

0.41 M.04

0.51 H.14

0.21 +0.08**

< 0.05 compared to group A :* p p < 0.001 compared to group A Amniotic fluid Amniotic fluid PG levels rose with advancing gestational age but this was less pronounced for PGF and PGFM than for PGE (Figure 1). PGE levels were higher than PGF.

36 (n=ll) Length of gestation bedcs)

Figure 1.

Amniotic fluid concentration of prostaglandins before labour. (Vertical bars represent mean plus standard error of mean). 313

There was no significant difference between amniotic sac concentrations of twin I and II in the prelabour group but all four patients in active labour with intact membranes had elevated PG levels in the first sac. There was a significant association between fetal plasma and amniotic fluid levels of PGE and PGFM in the prelabour group (n = 20, r = 0.56 and 0.57 respectively; p < 0.01). DISCUSSION The present study confirms that the umbilical vein of the fetus at term contains much higher concentrations of prostaglandins than are generally accepted for maternal plasma at this time (6,9). Although there are quite wide variations in absolute concentrations, the mean for twins reported here are similar to those recorded by Bibby -et al. (9) for singletons at elective Caesarean section. In particular we confirm the higher concentrations of PGE over PGF in the fetal circulation. No significant difference was found between prostaglandin levels in venous blood of twins growing in the same uterus. Several authors have noted the behaviour of prostaglandin concentrations in cord blood during the course of labour. Evidence presented here clearly indicates that there was no increase in prostaglandins during the latent phase of labour but that during active labour, PGE andPGFM were significantly increased over prelabour levels. Inasmuch as PGFM is the direct metabolite of the unstable compound PGF, it may be that this observation reflects a rise in both PGE and PGF during active labour although cord blood levels of the latter do not change significantly until delivery. There are several possible interpretations of this finding. Firstly, release of prostaglandins from the placenta due to uterine contractions may be reflected in the cord blood measurements. Alternatively, the rise could be a response to the greater physical stress to which the fetus is subjected during active labour. Determination of the origin of fetal prostaglandins has proved difficult as only one group has demonstrated an arteriovenous difference for the umbilical circulation (lo), and that only for PGE. Amniotic fluid concentrations of arachidonic acid derivatives have been assumed to be derived from the decidua and membranes and this is almost certainly true in active labour. However, we have demonstrated good correlation between fetal and amniotic fluid prostaglandins before labour (certainly for PGE and PGFM), suggesting either a common origin for both or a marked contribution to amniotic fluid via the fetal urine. Increasing amniotic fluid levels of PGE as gestation progresses follows a parallel increase in PGE concentration in the blood. The obvious question which arises from these studies is whether the prostaglandin increases are the consequences of endocrine maturity in the fetus with secondary effects on the initiation of labour, or whether the fetal endocrine svstem is resuondino to a maternal sictnal et al. (llj indicating that gestation has"run its course. Challis -have observed a rise in PGE 24 hrs before the onset of contractions in fetal lambs and noted that this followed the rise in cortisol which

314

has been implicated as the critical initiating feature of the onset of labour in this species. Louis et al. (13) and Pather -et al. (14) have shown that infusion of PGE into chronically catheterized fetal lambs elevates plasma ACTH and cortisol suggesting that changes in blood levels of PGE may affect adrenal secretion of glucocorticoids via the hypothalamic pituitary axis. Extrapolation of these observations to the human fetus is difficult as the initiation of labour is significantly different from that of ovine pregnancy (15). However, the demonstration that PGE, PGF and PGFM are higher at vaginal delivery than at elective Caesarean section may be consistent with the hypothesis that fetal prostaglandins play a role in the onset of labour by the regulation of cortisol, which rises with labour. Alternatively, increased excretion of arachidonic acid derivatives in the urine may sensitize the decidium andmyometrium to other factors involved in the precipitation of labour. These questions are the subject of continuing study. ACKNOWLEDGEMENTS This work was supported by the Preclinical Diagnostic Chemistry Research Group of the South African Medical Research Council. The authors are grateful to Dr M. Marivate and the Department of Obstetrics and Gynaecology for the use of their facilities. REFERENCES 1.

Challis, J.R.G., The production of prostaglanand Patrick, J.E. dins and thromboxanes in the feto-placental unit and their effects on the developing fetus. Semin. Perinatol. -4:23, 1980.

2.

Friedman. W.F., and Fitzoatrick, K.M. Effects of DrOStaqlandinS, thromboxanes and inhibitors of their synthesis on renal and gastrointestina1 function in the newborn period. Semin. Perinatol 4:143, 1980.

3.

iankin, J.H.G., and McLaughlin, M.K. The regulation of placental blood flows. J Develop. Physiol. -1:3, 1979.

4.

Pace-Aschiak, C .R. Developmental aspects of prostaglandin bio~. . synthetic and catabolic systems. Semin. Perinatol. -4:15, 1980.

5.

Dray, F., Charbonnel, B., and Maclouf, J. Radioimmunoassay of prostaglandins FCZ,El and E2 in human plasma. Europ. J. Clin. Invest. 5:311, 1975.

6.

Mitchell: M.D., Flint, A.P., Bibby, J., Brunt, J., Arnold, J.M., Anderson, A.B.M., and Turnbull, A.C. Plasma concentrations of prostaglandins during late human labour : influence of normal and preterm labour. J. Clin. Endocrinol. Metab. -46:947, 1978.

7.

Dray, F., and Frydman, R. Primary prostaglandins in amniotic fluid in pregnancy and spontaneous labour. Am. J. Obstet. Gynecol 126:13, 1976.

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8.

Challis, J.R.G., Osathanondh, R., Ryan, K.J., and Tulchinsky, D. Maternal and fetal plasma prostaglandin levels at vaginal delivery and Caesarean section. Prostaglandins 6:281, 1974.

9.

Bibby, J.G., Brunt, J.D., Hodgson, H., Mitchell, M.D., Anderson, A.B.M., and Turnbull, A.C. Prostaglandins in umbilical plasma at elective Caesarean section. Br. J. Obstet. Gynaecol. -86:282, 1979.

10.

Mitchell, M.D., Brunt, J., Bibby, J., Flint, A.P.F., Anderson, A. B.M., and Turnbull, A.C. Prostaglandins in the human umbilical circulation at birth. Br. J. Obstet. Gynaecol. -85:114, 1978.

11.

Granstrom, E., and Kindahl, H. Radioimmunoassay of prostaglandins and thromboxanes, p. 119 in Advances in Prostaglandin and Thromboxane Research, Vol. 5, ed. Frolich, J.C. Raven Press, New York, 1978.

12.

Challis, J.R.G., Dilley, S.R., Robinson, J.S., and Thoburn, G.D. Prostaglandins in the circulation of the fetal lamb. Prostaglandins -11:1041, 1976.

13.

Robinson, J.S., and Thoburn, G.D. Louis, T.M., Challis, J.R.G., Rapid increase of foetal corticosteroids after prostaglandin E2. Nature 264:79, 1976.

14.

Ratter, S., Rees, L.H., Landon, J.R. et al. The effect of prostaglandin E2 infusion in the fetal lamb on fetal plasma ACTH, prolactin, and cortisol concentrations. Prostaglandins -18:101, 1979.

15.

Turnbull, A.C., Anderson, A.B.M., Funt, A.P.F. et fl. Human parturition, p. 427 in The fetus and birth. CiE Foundation Symposium 47 (new series). Elsevier, Excerpta Medica, Oxford. 1979.

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