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Specific production of prostaglandin E by human amnion in vitro
PROSTAGLANDINS
SPECIFIC PRODUCTION OF PROSTAGLANDIN E BY hWJMAN AMNION IN VITRO M.D. Mitchell, J. Bibby,
B.R. Hicks and A.C. Turnbull
N u f f i e l d Department of Obstetrics and Gynaeoology, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9INJ, U.K. ABSTRACT Prostaglandin production by intra-uterine human tissues has been i n v e s t i g a t e d using a m e t h o d of tissue superfusion. Tissues were obtained at elective Caesarean section and after spontaneous vaginal delivery. It was found that all the tissues studied (amnion, chorion, decidua and placenta) p r o d u c e d more p r o s t a g l a n d i n E (PGE) and 13,14-dihydro-15-ketoprostaglandin F (PGFM - the major circulating metabolite of p r o s t a g l a n d i n F) than p r o s t a g l a n d i n F (PGF). Amnion p r o d u c e d significantly more P G E (but not PGF or PGFM) than any other tissue. Prostaglandin production by each tissue was similar whether it was taken at elective Caesarean section or after spontaneous vaginal delivery. INTRODUCTION There is a considerable body of evidence implicating prostaglandins in the mechanism(s) of parturition in many species (1,2). In man, there is an increase in the concentrations of prostaglandins in amniotic fluid ( 3 , 4 ) p l a s m a (5) and urine (6) during late pregnancy and labour. The source of these prostaglandins has remained controversial with the decidua (3,7), fetal membranes (8,9) and myometrium (lO) all having been suggested as possibilities. Interpretation of such data must, however, be cautious since tissue trauma (e.g. excision) results in the rapid synthesis and release of prostaglandins (ii). Another p r o b l e m is that the conversion of exogenous arachidonic acid to prostaglandin E (PGE) and prostaglandin F (PGF) by these tissues is extremely low (9) and difficult to separate from non-enzymatic production due to autooxidation. Furthermore, a recent study (12) has demonstrated that amnion rapidly degenerates in vitro unless special incubation media are employed. We have developed a method for the continuous superfusion of tissue (13) whereby prostaglandins released by the initial trauma of excision and manipulation can be removed before samples are obtained under steady state conditions. This methodology has been applied to the
FEBRUARY 1978 VOL.'I5 NO. 2
377
PROSTAGLANDINS
determination of possible intra-uterine sources of prostaglandins during late human pregnancy and parturition. PATI~TS
AND METHODS
Placentae were obtained f r o m twenty women who had uncomplicated pregnancies. All delivered a single live-born healthy infant at term. Ten women were delivered vaginally after labour which was of spontaneous onset and during which epidural anaesthesia was not administered. Another ten women were delivered by elective Caesarean section because of a presentation by the breech, cephalo-pelvic disproportion or a previous Caesarean section. Baby and placental weights were in the ranges 2,850-4050 g and 4 0 5 - 7 2 0 g respectively. Superfusion of tissue was carried out essentially as described p r e v i o u s l y (13). Tissues were rapidly minced with scissors and held between membrane filters in a Millipore holder within a bath at 37°C. A m o d i f i e d Krebs bicarbonate buffer containing glucose (2g/l) and bovine serum albumin (ig/l) was used to superfuse the tissue at a rate of 0.6 ml/min. A wash-out p e r i o d of 90 min was sufficient to eliminate prostaglandins released by initial trauma and p r o s t a g l a n d i n production was stable for up to I0 hr thereafter. The coefficient of variation of production by samples during this p e r i o d was 26~; this includes weighing and assay errors as well as possible natural fluctuations in production. Analysis of prostaglandin production by 18 duplicate samples of the same tissue revealed no statistical difference between productions (p>0.1). Superfusion of tissues with the modified Krebs bicarbonate buffer resulted in similar production rates of prostaglandins when compared to superfusion of duplicate tissues with the pseudoamniotic fluid described by Schwartz and co-workers (12). (amnion - t=36, n=12, p>0.1; chorion - t=6, n=8, p>O.l; decidua - t=26, n=12, p>O.l). In all cases super/usion of tissue started within 30 min of delivery and final dry weights of tissues were>100 mg. At least four samples of superfusate from each tissue were analysed for prostaglandin content by radioimmunoassays previously described (13). The inclusion of a chromatographic step in the assays was without effect on the concentrations of prostaglandins measured (t=36, n=16, p>O.l) and therefore chromatography was not routinely applied. During these experiments solvent blanks were
378
FEBRUARY 1978 VOL. 15 NO. 2
PROSTAGLANDINS
RESULTS There was no statistical difference (p>O.l) between prostaglandin production by tissues obtained after spontaneous vaginal delivery (Table I) and at elective Caesarean section (Table II). TABLE I Prostaglandin Production by Tissues obtained after Spontaneous Vaginal Delivery
TISSUE
Prostaglandin production ng/9. dry wt/min (mean - S.E.M.; n=lO) PGE
Am_nion
PGF
PGFM
13.17 (2.21)
0.83 (0.19)
1.43 (0.27)
Chorion
2.89 (0.46)
0.51 (0.12)
2.61 (0.71)
Decidua
1.72 (0.24)
0.49 (0.09)
1.51 (0.33)
Placenta
2.02 (0.38)
0.66 (O.13)
1.96 (0.29)
TABLE II Prostaglandin Production by Tissues obtained at Elective Caesarean Section
TISSUE
Prostaglandin production ng/g. dry wt/min (mean - S.E.M.; n=lO) PGE
PGF
PGFM
Amnion
9.62 (1.62)
0.74 (0.19)
0.94 (0.20)
Chorion
3.13 (0.59)
0.76 (0.20)
2.35 (0.41)
Decidua
2.50 (0.57)
0.80 (0.25)
2.06 (0.57)
Placenta
2.84 (0.47)
0.82 (0.24)
1.73 (0.32)
FEBRUARY 1978 VOL. 15 NO. 2
379
PROSTAGLANDINS
In tissues obtained both after spontaneous vaginal delivery and at elective Caesarean section the production of P G E and PGFM was consistently greater than the production of PGF (p
380
FEBRUARY 1978 VOL. 15 NO. 2
PROSTAGLANDINS
Alternatively prostaglandin synthetase activity may remain low during pregnancy (perhaps after inhibition during early pregnancy) until stimulated during late pregnancy at the onset of labour or by the trauma of Caesarean section. Interestingly suppression of prostaglandin synthesis has already been demonstrated during early pregnancy in the sheep (16) . The present results suggest that a significant potential for prostaglandin synthesis and metabolism has developed by late gestation in man and that labour per s_~e is without effect on this potential. The factors involved in the regulation of prostaglandin production in vivo remain to be established.
ACKNOWLEDGEMENTS We thank Dr. K. T. Kirton (Upjohn Co.,) for gifts of prostaglandin antisera and authentic standards and Dr. Mo Withnall (May and Baker Co.,) for the gift of an antiserum to PGE0 M.D°M. acknowledges receipt of the Staines Medical Research Fellowship (Exeter College, Oxford)° This work was supported by an M.R.C. Programme Grant awarded to A.C .T. REFERE/qCES
i.
Flint, A.P.F. and Hiller, K. In: Prostaglandins Reproduction. (S.MoMo Karim, ed). M.T.P. Press, Lancaster, 1976, p 27.
2.
Thorburn, G.D., Challis, JoR.G. and Currie, W.B. In: Biology of the Uterus. (R.M. Wynn, ed) Plenum Press, New York, 1977, p 653.
3.
Karim, S.M.M. and Devlin, J. Prostaglandin content of amniotic fluid during pregnancy and labour. J. Obstet. Gynaecol. Bro Commonw. 74; 230, 1967.
4.
Keirse, M.JoNoC° and Turnbull, A.C. E prostaglandins in amniotic fluid during late pregnancy and labour. Jo Obstet. Gynaecolo Bro Commonwo 80: 970, 1973.
5.
Green, K., Bygdeman, M., Toppozada, Mo and Wiqvist, N. The role of prostaglandin F2_ in human parturition. Endogenous plasma levels of ~5-keto-13,14-dihydroprostaglandin F_ during labor. Am° J. Obstet. Gynecol. 120: 25, 19 7 4. ~u
6.
Hamberg, M. Quantitative studies on prostaglandin synthesis in man. III. Excretion of the major urinary metabolites of prostaglandins F. and F_ during pregnancy. Life Sciences 14: 2 ~ , 1974~.a
FEBRUARY 1978 VOL. 15 NO. 2
and
381
PROSTAGLANDINS
7.
Gustavii, Bo In: The Fetus and Birth (J. Knight and M. O'Connor, eds) o Elsevier/Excerpta Medica/North Holland, AIqsterdam, 1977, p 343.
8.
MacDonald, P.C., Schultz, F.M., Duenhoelter, J.H.,Gant, N.~., Jimenez, J.M., Pritchard, J.A., Porter, J.C. and Johnston, J.Mo Initiation of human parturition. I. Mechanism of action of arachidonic acid. Obstet. Gynecol. 44: 629, 1974.
9.
Keirse, M.J.N.C. and Turnbull, A.C. The fetal membranes as a possible source of amniotic fluid prostaglandins. Br. J. Obstet. Gynaecol. 83: 146, 1976.
i0.
Willman, E.A. and Collins, W.P. Distribution of prostaglandins E_ and F~ within the foetoplacental unit throughout human pregnancy. J. Endocrinol. 69: 413, 1976.
iio
Piper, P.J. and Vane, J.R. The release of prostaglandins from lung and other tissues. Ann. N.Y. Acad. Sci. 1.80: 363, 1971
12.
Schwartz, A.L., F0rster, C.S., Smith, P.A. and Liggins, G.C. Human amnion metabolism. I. In vitro maintenance° Am. J. Obstet. Gynecol. 127: 470, 1977o
13.
Mitchell, M.D. and Flint, A.FoF. Prostaglandin production by intra-uterine tissues from periparturient sheep: use of a superfusion technique. J. Endocrinol. 7_~: iii, 1978.
14.
Mitchell, M.D., Sots, H. and Flint, A.P.F. Instability of 13,14-dihydro-15-keto-prostaglandin E 2. Lancet ~: 558) 1977.
]5.
Keirse, M.J.N.C. and Turnbull, A.C. Metabolism of prostaglandins within the pregnant uterus. Br. J. Obstet. Gynaecol. 82, 887, 1975.
16.
Thorburn, G.D., Cox, R.I., Currie, W.B., Restall, B.J. and Schneider, W. Prostaglandin F and progesterone concentrations in the utero-ovarian venous plasma of the ewe during the oestrous cycle and early pregnancy. J. Reprod. Fertil. supple 18, 151, 1973.
382
FEBRUARY 1978 VOL. 15 NO. 2
SPECIFIC PRODUCTION OF PROSTAGLANDIN E BY hWJMAN AMNION IN VITRO M.D. Mitchell, J. Bibby,
B.R. Hicks and A.C. Turnbull
N u f f i e l d Department of Obstetrics and Gynaeoology, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9INJ, U.K. ABSTRACT Prostaglandin production by intra-uterine human tissues has been i n v e s t i g a t e d using a m e t h o d of tissue superfusion. Tissues were obtained at elective Caesarean section and after spontaneous vaginal delivery. It was found that all the tissues studied (amnion, chorion, decidua and placenta) p r o d u c e d more p r o s t a g l a n d i n E (PGE) and 13,14-dihydro-15-ketoprostaglandin F (PGFM - the major circulating metabolite of p r o s t a g l a n d i n F) than p r o s t a g l a n d i n F (PGF). Amnion p r o d u c e d significantly more P G E (but not PGF or PGFM) than any other tissue. Prostaglandin production by each tissue was similar whether it was taken at elective Caesarean section or after spontaneous vaginal delivery. INTRODUCTION There is a considerable body of evidence implicating prostaglandins in the mechanism(s) of parturition in many species (1,2). In man, there is an increase in the concentrations of prostaglandins in amniotic fluid ( 3 , 4 ) p l a s m a (5) and urine (6) during late pregnancy and labour. The source of these prostaglandins has remained controversial with the decidua (3,7), fetal membranes (8,9) and myometrium (lO) all having been suggested as possibilities. Interpretation of such data must, however, be cautious since tissue trauma (e.g. excision) results in the rapid synthesis and release of prostaglandins (ii). Another p r o b l e m is that the conversion of exogenous arachidonic acid to prostaglandin E (PGE) and prostaglandin F (PGF) by these tissues is extremely low (9) and difficult to separate from non-enzymatic production due to autooxidation. Furthermore, a recent study (12) has demonstrated that amnion rapidly degenerates in vitro unless special incubation media are employed. We have developed a method for the continuous superfusion of tissue (13) whereby prostaglandins released by the initial trauma of excision and manipulation can be removed before samples are obtained under steady state conditions. This methodology has been applied to the
FEBRUARY 1978 VOL.'I5 NO. 2
377
PROSTAGLANDINS
determination of possible intra-uterine sources of prostaglandins during late human pregnancy and parturition. PATI~TS
AND METHODS
Placentae were obtained f r o m twenty women who had uncomplicated pregnancies. All delivered a single live-born healthy infant at term. Ten women were delivered vaginally after labour which was of spontaneous onset and during which epidural anaesthesia was not administered. Another ten women were delivered by elective Caesarean section because of a presentation by the breech, cephalo-pelvic disproportion or a previous Caesarean section. Baby and placental weights were in the ranges 2,850-4050 g and 4 0 5 - 7 2 0 g respectively. Superfusion of tissue was carried out essentially as described p r e v i o u s l y (13). Tissues were rapidly minced with scissors and held between membrane filters in a Millipore holder within a bath at 37°C. A m o d i f i e d Krebs bicarbonate buffer containing glucose (2g/l) and bovine serum albumin (ig/l) was used to superfuse the tissue at a rate of 0.6 ml/min. A wash-out p e r i o d of 90 min was sufficient to eliminate prostaglandins released by initial trauma and p r o s t a g l a n d i n production was stable for up to I0 hr thereafter. The coefficient of variation of production by samples during this p e r i o d was 26~; this includes weighing and assay errors as well as possible natural fluctuations in production. Analysis of prostaglandin production by 18 duplicate samples of the same tissue revealed no statistical difference between productions (p>0.1). Superfusion of tissues with the modified Krebs bicarbonate buffer resulted in similar production rates of prostaglandins when compared to superfusion of duplicate tissues with the pseudoamniotic fluid described by Schwartz and co-workers (12). (amnion - t=36, n=12, p>0.1; chorion - t=6, n=8, p>O.l; decidua - t=26, n=12, p>O.l). In all cases super/usion of tissue started within 30 min of delivery and final dry weights of tissues were>100 mg. At least four samples of superfusate from each tissue were analysed for prostaglandin content by radioimmunoassays previously described (13). The inclusion of a chromatographic step in the assays was without effect on the concentrations of prostaglandins measured (t=36, n=16, p>O.l) and therefore chromatography was not routinely applied. During these experiments solvent blanks were
378
FEBRUARY 1978 VOL. 15 NO. 2
PROSTAGLANDINS
RESULTS There was no statistical difference (p>O.l) between prostaglandin production by tissues obtained after spontaneous vaginal delivery (Table I) and at elective Caesarean section (Table II). TABLE I Prostaglandin Production by Tissues obtained after Spontaneous Vaginal Delivery
TISSUE
Prostaglandin production ng/9. dry wt/min (mean - S.E.M.; n=lO) PGE
Am_nion
PGF
PGFM
13.17 (2.21)
0.83 (0.19)
1.43 (0.27)
Chorion
2.89 (0.46)
0.51 (0.12)
2.61 (0.71)
Decidua
1.72 (0.24)
0.49 (0.09)
1.51 (0.33)
Placenta
2.02 (0.38)
0.66 (O.13)
1.96 (0.29)
TABLE II Prostaglandin Production by Tissues obtained at Elective Caesarean Section
TISSUE
Prostaglandin production ng/g. dry wt/min (mean - S.E.M.; n=lO) PGE
PGF
PGFM
Amnion
9.62 (1.62)
0.74 (0.19)
0.94 (0.20)
Chorion
3.13 (0.59)
0.76 (0.20)
2.35 (0.41)
Decidua
2.50 (0.57)
0.80 (0.25)
2.06 (0.57)
Placenta
2.84 (0.47)
0.82 (0.24)
1.73 (0.32)
FEBRUARY 1978 VOL. 15 NO. 2
379
PROSTAGLANDINS
In tissues obtained both after spontaneous vaginal delivery and at elective Caesarean section the production of P G E and PGFM was consistently greater than the production of PGF (p
380
FEBRUARY 1978 VOL. 15 NO. 2
PROSTAGLANDINS
Alternatively prostaglandin synthetase activity may remain low during pregnancy (perhaps after inhibition during early pregnancy) until stimulated during late pregnancy at the onset of labour or by the trauma of Caesarean section. Interestingly suppression of prostaglandin synthesis has already been demonstrated during early pregnancy in the sheep (16) . The present results suggest that a significant potential for prostaglandin synthesis and metabolism has developed by late gestation in man and that labour per s_~e is without effect on this potential. The factors involved in the regulation of prostaglandin production in vivo remain to be established.
ACKNOWLEDGEMENTS We thank Dr. K. T. Kirton (Upjohn Co.,) for gifts of prostaglandin antisera and authentic standards and Dr. Mo Withnall (May and Baker Co.,) for the gift of an antiserum to PGE0 M.D°M. acknowledges receipt of the Staines Medical Research Fellowship (Exeter College, Oxford)° This work was supported by an M.R.C. Programme Grant awarded to A.C .T. REFERE/qCES
i.
Flint, A.P.F. and Hiller, K. In: Prostaglandins Reproduction. (S.MoMo Karim, ed). M.T.P. Press, Lancaster, 1976, p 27.
2.
Thorburn, G.D., Challis, JoR.G. and Currie, W.B. In: Biology of the Uterus. (R.M. Wynn, ed) Plenum Press, New York, 1977, p 653.
3.
Karim, S.M.M. and Devlin, J. Prostaglandin content of amniotic fluid during pregnancy and labour. J. Obstet. Gynaecol. Bro Commonw. 74; 230, 1967.
4.
Keirse, M.JoNoC° and Turnbull, A.C. E prostaglandins in amniotic fluid during late pregnancy and labour. Jo Obstet. Gynaecolo Bro Commonwo 80: 970, 1973.
5.
Green, K., Bygdeman, M., Toppozada, Mo and Wiqvist, N. The role of prostaglandin F2_ in human parturition. Endogenous plasma levels of ~5-keto-13,14-dihydroprostaglandin F_ during labor. Am° J. Obstet. Gynecol. 120: 25, 19 7 4. ~u
6.
Hamberg, M. Quantitative studies on prostaglandin synthesis in man. III. Excretion of the major urinary metabolites of prostaglandins F. and F_ during pregnancy. Life Sciences 14: 2 ~ , 1974~.a
FEBRUARY 1978 VOL. 15 NO. 2
and
381
PROSTAGLANDINS
7.
Gustavii, Bo In: The Fetus and Birth (J. Knight and M. O'Connor, eds) o Elsevier/Excerpta Medica/North Holland, AIqsterdam, 1977, p 343.
8.
MacDonald, P.C., Schultz, F.M., Duenhoelter, J.H.,Gant, N.~., Jimenez, J.M., Pritchard, J.A., Porter, J.C. and Johnston, J.Mo Initiation of human parturition. I. Mechanism of action of arachidonic acid. Obstet. Gynecol. 44: 629, 1974.
9.
Keirse, M.J.N.C. and Turnbull, A.C. The fetal membranes as a possible source of amniotic fluid prostaglandins. Br. J. Obstet. Gynaecol. 83: 146, 1976.
i0.
Willman, E.A. and Collins, W.P. Distribution of prostaglandins E_ and F~ within the foetoplacental unit throughout human pregnancy. J. Endocrinol. 69: 413, 1976.
iio
Piper, P.J. and Vane, J.R. The release of prostaglandins from lung and other tissues. Ann. N.Y. Acad. Sci. 1.80: 363, 1971
12.
Schwartz, A.L., F0rster, C.S., Smith, P.A. and Liggins, G.C. Human amnion metabolism. I. In vitro maintenance° Am. J. Obstet. Gynecol. 127: 470, 1977o
13.
Mitchell, M.D. and Flint, A.FoF. Prostaglandin production by intra-uterine tissues from periparturient sheep: use of a superfusion technique. J. Endocrinol. 7_~: iii, 1978.
14.
Mitchell, M.D., Sots, H. and Flint, A.P.F. Instability of 13,14-dihydro-15-keto-prostaglandin E 2. Lancet ~: 558) 1977.
]5.
Keirse, M.J.N.C. and Turnbull, A.C. Metabolism of prostaglandins within the pregnant uterus. Br. J. Obstet. Gynaecol. 82, 887, 1975.
16.
Thorburn, G.D., Cox, R.I., Currie, W.B., Restall, B.J. and Schneider, W. Prostaglandin F and progesterone concentrations in the utero-ovarian venous plasma of the ewe during the oestrous cycle and early pregnancy. J. Reprod. Fertil. supple 18, 151, 1973.
382
FEBRUARY 1978 VOL. 15 NO. 2