Department of Obstetrics King's College Hospital Denmark Hill,
and Gynaecology, Medical School, London S. E. 5
THE CONCENTRATION OF PROSTAGLANDIN F2\g=a\ IN MATERNAL PLASMA, FOETAL PLASMA
AND AMNIOTIC FLUID DURING PREGNANCY IN WOMEN
D. A. Johnson, P. A. Manning, J. F. Hennam, J. R. Newton and W. P. Collins ABSTRACT The concentration of
prostaglandin F2\g=a\ has been determined in serial samples of peripheral venous plasma from women at defined times during labour, and studied in detail throughout two consecutive uterine contractions. In addition, the same compound has been measured in single samples of uterine venous plasma, cord venous plasma, and amniotic fluid in groups of patients during early and late pregnancy, labour and at delivery of the baby. The results from the analysis of peripheral venous plasma show that there is considerable individual variation in the concentration of prosta-
glandin F2\g=a\during labour (mean \m=+-\sd, 33.1 \m=+-\11.6 pg/ml). However, it is not possible to establish a definite correlation with either the latent or accelerated phases or with the time of delivery. Furthermore, there is no apparent temporal relationship between the concentrations in peripheral venous plasma and the contractile state of the uterus as assessed by external tocography. In early pregnancy (16th to 20th week) the concentration of prostaglandin F2\g=a\(pg/ml, mean \m=+-\sd) in peripheral venous plasma is 26.3 \m=+-\4.3 and in amniotic fluid 32.7 \m=+-\26.5. At the 36th week to the start of labour the corresponding values are 27.1 \m=+-\8.1 and 110.0 \m=+-\73.8. At the same time the levels in cord plasma and uterine venous plasma are 100.4 \m=+-\74.9 and 87.9 \m=+-\55.0 respectively. During labour there is a significant in-
(P < 0.005, Student's t-test) in the concentration in amniotic fluid \m=+-\171.0). The results are discussed in relation to the possible role of prostaglandin F2\g=a\in the process of parturition. crease
(335.1
It is well known that the administration of various prostaglandins to pregnant women causes an increase in the frequency and amplitude of uterine con¬ tractions (Karim 1972). Furthermore, the results from numerous studies suggest that prostaglandin F2a is involved in the initiation of parturition in some labo¬ ratory and domestic animals (Caldwell et al. 1973). However, the role of naturally occurring prostaglandins in the regulation of labour in women is not so well established. The relevant studies have been hampered by the inacces¬ sibility of the foeto-placental unit, and by the low concentration and rapid metabolism of the active compounds. The present communication is concerned with the concentration of prosta¬ glandin Fga in peripheral venous plasma at defined times during labour, and in serial samples throughout two consecutive uterine contractions. In addition, a comparison has been made between the relative levels of this compound in uterine venous plasma, cord plasma and amniotic fluid at selected times during
gestation. EXPERIMENTAL
Subjects Samples of plasma and amniotic fluid were obtained from patients between the 16th and 20th week of pregnancy immediately prior to termination for socio-economic reasons. Corresponding samples were obtained from patients during late pregnancy or labour, at elective or emergency Caesarian section respectively. In addition, speci¬ mens of amniotic fluid, peripheral vein and cord plasma were obtained from patients who had a spontaneous labour and delivery. Materials An authentic sample of prostaglandin F2a was donated by the Ono Pharmaceutical Co. Ltd., Osaka, Japan, and [5, 6, 8, 9, 11, 12, 14, 15-H3]prostagandin Fo(( (specific activity 120 Cì/itvm) was purchased from the New England Nuclear Corp., Boston, Mass., USA. An antiserum to prostaglandin F2a-l-bovine serum albumin was raised in rabbits, and under the conditions of the assay, the maximum cross-reaction with related compounds that were available for testing was with prostaglandin F%a
(5.1
per
cent).
Method The concentration of prostaglandin F¡u was determined in all samples by a method based upon the principles of radioimmunoassay. Details of the procedure and a com¬ plete evaluation for application to samples of peripheral venous plasma has been published (Hennam et al. 1974). The salient features of the method as applied to the
various fluids analysed in the present study are as follows: Immediately after blood is withdrawn, the sample is transferred to a lithium heparin tube, and the plasma separated by centrifugation at 10°C. If possible 2 ml aliquots are removed in duplicate for assay, together with similar volumes of amniotic fluid. Two thousand dpm of tritiated prostaglandin F2tt (equivalent to about 2 pg) in 100 µ of water is added, followed by 200 µ of normal hydrochloric acid. The contents are mixed, and the sample may be stored up to 24 h at 4°C. The prostaglandins are extracted with freshly distilled diethyl ether, once with 10 ml and then with 5 ml. The pooled extracts are dried under nitrogen at 40°C, and subsequently re-dissolved in 500 pi of buffer. Duplicate 100 µ samples are taken for assay, and the radioactivity in 200 µ is determined so that the losses may be calculated. After equilibration with suitably diluted antiserum (30 min at 4°C), and 20 000 dpm of tritiated prostaglandin F?a (an additional 30 min at 4°C), the antibody bound material is precipitated by the addition of 1 ml of a mixture of ammonium and calcium sulphate (ACS) at pH 8. This reagent contains 65 per cent saturated ammonium sulphate solution, and a suspen¬ sion of 40 mg of powdered calcium sulphate dihydrate. After centrifugation (6°C, 2000 g, 10 min), the supernatant is discarded and the precipitate re-suspended in 400 «1 of water. One ml of scintillation fluid is added to each tube. The tubes are capped and the contents mixed. The tubes are placed in counting vials which are transferred to a liquid scintillation counter, and the absolute amount of radioactivity is determined in every sample by a channels ratio method. A standard curve is constructed after analysing several samples containing known amounts of prostaglandin Foa, and plotting the mass on the abscissa against radio¬ activity bound on the ordinate. The mass in each biological extract is determined, and the value corrected for experimental losses, the aliquot taken for assay, and the initial volume of the sample. A summary of the evaluation is shown in Table 1. The accuracy refers to the deter¬ mination of various amounts of prostaglandin Fa^ added to samples or deionized, dis¬ tilled water over the range 0 to 300 pg/ml. The high degree of accuracy in these
Table 1. An evaluation of
a
radioimmunological procedure of prostaglandin F2a.
Parameter
Int. std.
ree.
(°/o,
mean
for the determination
Value
± sd)
Prostaglandin equivalent of blank (pg/ml, mean + sd) Precision (coeffic. of var. %>)
93 + 8
5 ± 2
Intra-assay Inter-assay
Accuracy (recovery, mean ± sd) Sensitivity (value significantly different from method blank, pg/ml)
8 14 95 + 6
10
determinations is indicative of similar binding characteristics for both the authentic and previously measured material. Furthermore, the attainment of consistently low blank values, and an increase in the basal levels of prostaglandin F2„ following the infusion of authentic material is additional evidence of specificity for the procedure as applied to the various fluids. The method for the determination of plasma progesterone is essentially similar to that described by Youssefnejadían et al. (1972). However, a more specific antiserum to progesterone-llct-succinyl-bovine serum albumin was used, in that the cross-reactions with 5a-dihydroprogesterone, 17-hydroxyprogesterone and 16ct-hydroxyprogesterone were all less than 10 per cent and the Chromatographie step was omitted.
RESULTS
Peripheral
venous
plasma:
serial
samples
The methods have been applied to the determination of prostaglandin F2a and progesterone, in serial samples of peripheral venous plasma removed at defined times from eight patients, in whom labour had been induced by low amniotomy and oxytocin titration. Samples of blood were removed from the contralateral antecubital vein by a separate venepuncture at the following times: 1. immediately prior to membrane rupture 2. during labour when the cervix was dilated six to 3. at delivery 4. one hour post-partum
eight
cm
A summary of the results is shown in Table 2. A statistical analysis of these values showed that the level of progesterone was significantly lower after de¬ livery of the foetus and placenta (P < 0.05, Student's i-test). An examination of the concentration of prostaglandin F2a in individual patients showed that although there were considerable fluctuations in the level, there was no conTable 2. The concentration venous
Prostaglandin F2a (pg/ml) Progesterone (ng/ml)
±
(mean plasma
sd) of from 8
prostaglandin F2a and progesterone in peripheral patients at defined stages of labour.
Membrane
Accelerated
rupture
phase
Delivery
1 h
post-partum
31.1 ± 11.1
31.5 ± 11.8
40.1 ± 17.6
35.9 ± 14.6
239 ± 75
221 ± 103
209 ± 79
139 ± 69
sistent pattern. The corresponding values for progesterone showed that there was a gradual reduction in the concentration of this compound in 50 per cent of the subjects, and in the remainder there was no significant change until the baby had been delivered. In a further five subjects, the concentration of both compounds was deter¬ mined in serial samples of plasma, collected over 10 second intervals through¬ out two consecutive uterine contractions as monitored by external tocography (Hewlett Packard Model 8020A). All subjects were in the late first stage of labour, when the uterine contractions were lasting from 45 to 55 seconds and were occurring every 2 to 3 min. The blood was withdrawn from an antecubital vein via an intravenous plastic cannula (Angiocath 1968). A detailed examination of the results from every patient showed that there
Group
Total No. of samples
(1) (2) (3) (4) (5)
36 32 37 35 36
venous
33.7 ± 9.9 35.4 ± 12.6 34.7 ± 10.0 34.4 ± 10.8 36.1 ± 12.0
Progesterone (ng/ml mean + sd) 210 203 214 207 211
± 65 ± 69 ± 67 ± 68 ±66
Fig- 1. prostaglandin Fga and progesterone in serial samples of plasma (10 seconds intervals) throughout 2 uterine contractions in 5 patients.
The concentrations of
peripheral
Prostaglandin Foa (pg/ml mean + sd)
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and Gynaecology, Medical School, London S. E. 5
THE CONCENTRATION OF PROSTAGLANDIN F2\g=a\ IN MATERNAL PLASMA, FOETAL PLASMA
AND AMNIOTIC FLUID DURING PREGNANCY IN WOMEN
D. A. Johnson, P. A. Manning, J. F. Hennam, J. R. Newton and W. P. Collins ABSTRACT The concentration of
prostaglandin F2\g=a\ has been determined in serial samples of peripheral venous plasma from women at defined times during labour, and studied in detail throughout two consecutive uterine contractions. In addition, the same compound has been measured in single samples of uterine venous plasma, cord venous plasma, and amniotic fluid in groups of patients during early and late pregnancy, labour and at delivery of the baby. The results from the analysis of peripheral venous plasma show that there is considerable individual variation in the concentration of prosta-
glandin F2\g=a\during labour (mean \m=+-\sd, 33.1 \m=+-\11.6 pg/ml). However, it is not possible to establish a definite correlation with either the latent or accelerated phases or with the time of delivery. Furthermore, there is no apparent temporal relationship between the concentrations in peripheral venous plasma and the contractile state of the uterus as assessed by external tocography. In early pregnancy (16th to 20th week) the concentration of prostaglandin F2\g=a\(pg/ml, mean \m=+-\sd) in peripheral venous plasma is 26.3 \m=+-\4.3 and in amniotic fluid 32.7 \m=+-\26.5. At the 36th week to the start of labour the corresponding values are 27.1 \m=+-\8.1 and 110.0 \m=+-\73.8. At the same time the levels in cord plasma and uterine venous plasma are 100.4 \m=+-\74.9 and 87.9 \m=+-\55.0 respectively. During labour there is a significant in-
(P < 0.005, Student's t-test) in the concentration in amniotic fluid \m=+-\171.0). The results are discussed in relation to the possible role of prostaglandin F2\g=a\in the process of parturition. crease
(335.1
It is well known that the administration of various prostaglandins to pregnant women causes an increase in the frequency and amplitude of uterine con¬ tractions (Karim 1972). Furthermore, the results from numerous studies suggest that prostaglandin F2a is involved in the initiation of parturition in some labo¬ ratory and domestic animals (Caldwell et al. 1973). However, the role of naturally occurring prostaglandins in the regulation of labour in women is not so well established. The relevant studies have been hampered by the inacces¬ sibility of the foeto-placental unit, and by the low concentration and rapid metabolism of the active compounds. The present communication is concerned with the concentration of prosta¬ glandin Fga in peripheral venous plasma at defined times during labour, and in serial samples throughout two consecutive uterine contractions. In addition, a comparison has been made between the relative levels of this compound in uterine venous plasma, cord plasma and amniotic fluid at selected times during
gestation. EXPERIMENTAL
Subjects Samples of plasma and amniotic fluid were obtained from patients between the 16th and 20th week of pregnancy immediately prior to termination for socio-economic reasons. Corresponding samples were obtained from patients during late pregnancy or labour, at elective or emergency Caesarian section respectively. In addition, speci¬ mens of amniotic fluid, peripheral vein and cord plasma were obtained from patients who had a spontaneous labour and delivery. Materials An authentic sample of prostaglandin F2a was donated by the Ono Pharmaceutical Co. Ltd., Osaka, Japan, and [5, 6, 8, 9, 11, 12, 14, 15-H3]prostagandin Fo(( (specific activity 120 Cì/itvm) was purchased from the New England Nuclear Corp., Boston, Mass., USA. An antiserum to prostaglandin F2a-l-bovine serum albumin was raised in rabbits, and under the conditions of the assay, the maximum cross-reaction with related compounds that were available for testing was with prostaglandin F%a
(5.1
per
cent).
Method The concentration of prostaglandin F¡u was determined in all samples by a method based upon the principles of radioimmunoassay. Details of the procedure and a com¬ plete evaluation for application to samples of peripheral venous plasma has been published (Hennam et al. 1974). The salient features of the method as applied to the
various fluids analysed in the present study are as follows: Immediately after blood is withdrawn, the sample is transferred to a lithium heparin tube, and the plasma separated by centrifugation at 10°C. If possible 2 ml aliquots are removed in duplicate for assay, together with similar volumes of amniotic fluid. Two thousand dpm of tritiated prostaglandin F2tt (equivalent to about 2 pg) in 100 µ of water is added, followed by 200 µ of normal hydrochloric acid. The contents are mixed, and the sample may be stored up to 24 h at 4°C. The prostaglandins are extracted with freshly distilled diethyl ether, once with 10 ml and then with 5 ml. The pooled extracts are dried under nitrogen at 40°C, and subsequently re-dissolved in 500 pi of buffer. Duplicate 100 µ samples are taken for assay, and the radioactivity in 200 µ is determined so that the losses may be calculated. After equilibration with suitably diluted antiserum (30 min at 4°C), and 20 000 dpm of tritiated prostaglandin F?a (an additional 30 min at 4°C), the antibody bound material is precipitated by the addition of 1 ml of a mixture of ammonium and calcium sulphate (ACS) at pH 8. This reagent contains 65 per cent saturated ammonium sulphate solution, and a suspen¬ sion of 40 mg of powdered calcium sulphate dihydrate. After centrifugation (6°C, 2000 g, 10 min), the supernatant is discarded and the precipitate re-suspended in 400 «1 of water. One ml of scintillation fluid is added to each tube. The tubes are capped and the contents mixed. The tubes are placed in counting vials which are transferred to a liquid scintillation counter, and the absolute amount of radioactivity is determined in every sample by a channels ratio method. A standard curve is constructed after analysing several samples containing known amounts of prostaglandin Foa, and plotting the mass on the abscissa against radio¬ activity bound on the ordinate. The mass in each biological extract is determined, and the value corrected for experimental losses, the aliquot taken for assay, and the initial volume of the sample. A summary of the evaluation is shown in Table 1. The accuracy refers to the deter¬ mination of various amounts of prostaglandin Fa^ added to samples or deionized, dis¬ tilled water over the range 0 to 300 pg/ml. The high degree of accuracy in these
Table 1. An evaluation of
a
radioimmunological procedure of prostaglandin F2a.
Parameter
Int. std.
ree.
(°/o,
mean
for the determination
Value
± sd)
Prostaglandin equivalent of blank (pg/ml, mean + sd) Precision (coeffic. of var. %>)
93 + 8
5 ± 2
Intra-assay Inter-assay
Accuracy (recovery, mean ± sd) Sensitivity (value significantly different from method blank, pg/ml)
8 14 95 + 6
10
determinations is indicative of similar binding characteristics for both the authentic and previously measured material. Furthermore, the attainment of consistently low blank values, and an increase in the basal levels of prostaglandin F2„ following the infusion of authentic material is additional evidence of specificity for the procedure as applied to the various fluids. The method for the determination of plasma progesterone is essentially similar to that described by Youssefnejadían et al. (1972). However, a more specific antiserum to progesterone-llct-succinyl-bovine serum albumin was used, in that the cross-reactions with 5a-dihydroprogesterone, 17-hydroxyprogesterone and 16ct-hydroxyprogesterone were all less than 10 per cent and the Chromatographie step was omitted.
RESULTS
Peripheral
venous
plasma:
serial
samples
The methods have been applied to the determination of prostaglandin F2a and progesterone, in serial samples of peripheral venous plasma removed at defined times from eight patients, in whom labour had been induced by low amniotomy and oxytocin titration. Samples of blood were removed from the contralateral antecubital vein by a separate venepuncture at the following times: 1. immediately prior to membrane rupture 2. during labour when the cervix was dilated six to 3. at delivery 4. one hour post-partum
eight
cm
A summary of the results is shown in Table 2. A statistical analysis of these values showed that the level of progesterone was significantly lower after de¬ livery of the foetus and placenta (P < 0.05, Student's i-test). An examination of the concentration of prostaglandin F2a in individual patients showed that although there were considerable fluctuations in the level, there was no conTable 2. The concentration venous
Prostaglandin F2a (pg/ml) Progesterone (ng/ml)
±
(mean plasma
sd) of from 8
prostaglandin F2a and progesterone in peripheral patients at defined stages of labour.
Membrane
Accelerated
rupture
phase
Delivery
1 h
post-partum
31.1 ± 11.1
31.5 ± 11.8
40.1 ± 17.6
35.9 ± 14.6
239 ± 75
221 ± 103
209 ± 79
139 ± 69
sistent pattern. The corresponding values for progesterone showed that there was a gradual reduction in the concentration of this compound in 50 per cent of the subjects, and in the remainder there was no significant change until the baby had been delivered. In a further five subjects, the concentration of both compounds was deter¬ mined in serial samples of plasma, collected over 10 second intervals through¬ out two consecutive uterine contractions as monitored by external tocography (Hewlett Packard Model 8020A). All subjects were in the late first stage of labour, when the uterine contractions were lasting from 45 to 55 seconds and were occurring every 2 to 3 min. The blood was withdrawn from an antecubital vein via an intravenous plastic cannula (Angiocath 1968). A detailed examination of the results from every patient showed that there
Group
Total No. of samples
(1) (2) (3) (4) (5)
36 32 37 35 36
venous
33.7 ± 9.9 35.4 ± 12.6 34.7 ± 10.0 34.4 ± 10.8 36.1 ± 12.0
Progesterone (ng/ml mean + sd) 210 203 214 207 211
± 65 ± 69 ± 67 ± 68 ±66
Fig- 1. prostaglandin Fga and progesterone in serial samples of plasma (10 seconds intervals) throughout 2 uterine contractions in 5 patients.
The concentrations of
peripheral
Prostaglandin Foa (pg/ml mean + sd)
+1 +1 -h
CM
CO
—I
Q
c ho
«1
o
-H +1 +1 +1 ho
C
—
R
3
Q
O
*
*
OO
CO O O 00
I-h! c
T
ca j¿ C eu ho ej > «J
S
IU
—r
ca
^H r—
CÓ líS r-. ir>
+1 +1 +1 +1
en!
CM
* es q O r^ o O CO —i
co
00
-
OO
00
CJ
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^
co CM
cq iq
co CM CM
.h
+1 +1 +1 +1 CO «I
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cu
ce
f-i
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ce
W
CM CO
ß
--
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s
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î1
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0h
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U X
