1 Institute of Obstetrics and Gynaecology, 2 Department of Dermatology, Bialystok Medical University, Poland, 3 Technical University, Bialystok, Poland and 4 Department of Obstetrics and Gynaecology, University Hospital, Lund, Sweden
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Abstract |
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Key words: calcium/fetal membranes/magnesium/permeability
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Introduction |
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It is recognized that fetal membranes may play a central role in paracrine signalling during the initiation of parturition. A variety of hormones, cytokines and growth factors produced by fetal membranes and placentae can act locally on the myometrium. Considerable evidence exists suggesting that prostaglandins synthesized by the fetal membranes play an important role in the activation of myometrium and initiation of parturition (Bennett et al., 1990; Gibb, 1998
).
Recently, a great deal of interest has been focused on the role of nitric oxide (NO) in the initiation of parturition at term and implications of NO in preterm labour (Sladek et al., 1997; Nanno et al., 1998
). Evidence that NO has an important role in myometrial function comes from studies showing NO generated by nitric oxide synthase (NOS) from arginine has a relaxant effect on the myometrium and this relaxation is specifically blocked by inhibitors of NOS (Buhimschi et al., 1995
; Yallampali et al., 1996). Interestingly, in addition to presence of NOS, a key enzyme for the production of NO from arginine, in the myometrium and placenta, recent work has demonstrated its existence in fetal membranes (Dennes et al., 1997
).
Increased production of prostaglandins (PG) is a common occurrence in the mechanism of parturition and is thought to play an important role in the modulation of uterine contractile activity and cervical ripening. Evidence is rapidly accumulating for the existence of an interaction between NO and the PG synthesizing enzyme cyclo-oxygenase in a variety of tissues. The ability of NO to enhance PG biosynthesis has been observed in a variety of cell cultures and in-vivo models (Davidge et al., 1995; Salvemini et al., 1995
). Evidence has also been presented showing that NO can modulate uterine contractility by means of PGE production (Chaud et al., 1997
; Motta et al., 1998
).
The activity of constitutive NOS is dependent on calcium (Ca2+; Knowles and Moncada, 1994) and is inhibited by a reduction in the concentration of magnesium (Mg2+; Pearson et al., 1998). Magnesium has been extensively used for the prevention of premature labour. Both cations but particularly Ca2+ are co-factors in the synthetic activity of a variety of enzymes and in the secretory process. Both binding to fetal membranes and diffusion through the membranes of these two inorganic constituents of fetal membranes and amniotic fluid could be important factors in the synthesis and/or action of PG and generation of NO which are believed to regulate myometrial activity particularly for the induction of labour.
Amnion and chorion are very permeable to water and semi-permeable to other substances. Small molecules such as sodium and glucose go through as a result of simple diffusion but fetal membranes do not readily permit substances of molecular weight >1000 Da to get across (Seeds, 1970). In view of the fact that chemical agents diffusing across the fetal membrane can affect the myometrial activity directly or indirectly through the synthesis of uterotonic or tocolytic agents, it is important to know whether the differences in permeability of fetal membranes exist in preterm and term deliveries.
Since no previous data are available on the diffusion of Ca2+ and Mg2+ through fetal membranes, we have in the present study examined the permeability to these cations of chorioamniotic membranes obtained from women who had undergone term or preterm labour.
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Materials and methods |
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This hourly withdrawal of 1 ml samples (16 samples in all) should have led to a steady decrease in the Ca2+ or Mg2+ concentration. However, due to the diffusion of solution from the transmission chamber, which initially contained a 10-fold higher concentration of Ca2+ and Mg2+, some increase in the concentration of these cations would be expected in the reception chamber. In order to account for these changes, a mathematical model was used to make a correction in the experimental data. The double compartment model (Godfrey, 1983) was modified to accommodate sample extractions.
Taking the following assumptions: (i) no chemical changes occur in the tested substances during transportation from one chamber to the other through the membrane, (ii) the total amount of substance in both chambers at any given time remains the same, (iii) the membrane that divides two chambers is so thin that it does not accumulate significant amounts of the tested substance and (iv) there are no convection movements in the tested substance, then the substance concentrations equations in the transmission and the reception chambers may be calculated as follows (Czy|ewska et al., 1997):
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where:
Ct, Cr = ion concentrations in the transmission and reception chambers;
K = transport coefficient between the two chambers (in h1);
B = the equilibrium concentration that could be measured in both chambers.
The sum At + B (Ar + B) is the concentration in the transmission (reception) chamber at the beginning of the experiment (t0):
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The model should be modified to accommodate sample extractions. Concentration change in the reception chamber as a result of sample extraction can be defined by the equation:
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where:
Vsum=volume of the extracted sample;
Vch=volume of chamber from which sample is extracted;
c2,i=concentration in the reception chamber at the end of the i th hour of the experiment.
Analysis of Ca2+ and Mg2+ in the samples was done with ion chromatography using a Metrohm IC-690 chromatograph fitted with Super SEP cation column and a Metrodata 714-IC version 2.06 computer program (Metrohm Ltd, Herisau, Switzerland).
Statistical analysis
Non-linear regression analysis with time as the independent variable was used. The assumptions of method were checked graphically and by using the KolmogorovSmirnov two-sample method. All the tests were performed using Prism version 2.01 (GraphPad Software Inc., San Diego, CA, USA).
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Results |
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To compare the permeability of Mg2+ and Ca2+ ions through preterm and term fetal membranes non-linear regression analysis was performed.
Based on the analysis above, a one-phase exponential decay curve was fitted to the data:
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The B coefficient was set as a constant and was computed as the average of ion concentrations in the transmission and the reception chambers at the beginning of the experiment minus the sample extraction adjustment according to equation 2 (Table II).
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To compare Ar and K coefficients the 0.05 level of significance was adopted (Table II). Differences between Ar coefficients for Ca2+ term and Ca2+ preterm as well as for Mg2+ term and Mg2+ preterm were not significant (Table II
: 95% CI for Ar).
The differences in K coefficients between term and preterm were significant for Mg2+ and Ca2+ ions (Table II: 95% CI for K).
Hence, permeability of Mg2+ and Ca2+ ions is different in the term and preterm fetal membranes.
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Discussion |
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In addition to a number of factors that may participate in the process of the initiation of labour, NO has recently been considered to play an important role in maintaining uterine quiescence (Yallampalli et al., 1993; Sladek et al., 1997). Since production of NO by constitutive NOS is calcium-dependent, one would expect a diminished formation of NO by the myometrium resulting from reduced availability of Ca2+ in preterm labour. However, compared with placenta, the concentration of calcium-dependent NOS in the pregnant myometrium is very low (Rasmay et al., 1996
; Sahin-Tóth et al., 1997
). We have recently found that the concentration of NOS in the placenta was 67-fold higher than in the myometrium obtained from the same patient (S.Batra, unpublished data). Consequently, it has been suggested that NO production by the myometrium is unlikely to play a role in uterine quiescence during pregnancy. On the other hand, due to a substantially higher concentration of NOS in placenta, it is highly likely that production of NO by placental NOS would be an important factor acting in a paracrine manner and contributing to uterine quiescence. Recent data in rats indicated that the presence of placental tissue enhanced inhibition of uterine contractility by agents that spontaneously released NO (Syal et al., 1999
).
A down-regulation of NOS activity resulting from diminished availability of Ca2+ in preterm labour would lower the inhibitory influence of the placental NOS-NO system on the myometrium and thereby contribute to the initiation of preterm labour. It is also possible that lower diffusion of Ca2+ through fetal membrane would lead to lower cellular uptake of this cation in the fetal membrane and result in diminished NOS activity in these membranes. Low Ca2+ and Mg2+ have previously been reported in prematurely ruptured membranes (PROM) compared with normal fetal membranes (Kidroni et al., 1989).
In a recent study it was suggested that NO synthesized in fetal membranes may act either directly to inhibit myometrial contractility or indirectly through an interaction with cyclo-oxygenase (Dennes et al., 1997; Motta et al., 1998
). This would also apply to NO produced by the placenta, which might be the major source of NO production during pregnancy.
Cations, particularly Ca2+ and Mg2+, are of crucial importance both in the physiology of pregnancy and in the pharmacotherapy of preterm labour. Both magnesium salts and calcium channel blockers have been used for suppression of preterm labour (Johnson, 1993). The mechanism by which Mg2+ helps to prevent preterm labour is not known; it is thought to have a membrane stabilizing action. Insight into the cellular mechanism underlying Mg2+ inhibition of myometrial contractions was provided previously (Popper et al., 1989
) showing a rapid and dose dependent inhibition of Ca2+ ions uptake in myometrial tissue obtained from pregnant women. The authors further showed that Mg2+ inhibited both spontaneous and k+-simulated myometrial contractions. A recent study (Phillipe, 1998) provides strong support for the conclusion that Mg2+ in relatively high concentrations inhibits extracellular Ca2+ entry (Philippe, 1998
). In a separate study (Batra and Popper, 1989
), a very high affinity of pregnant human myometrium was shown for the dihydropyridine calcium channel blockers (Batra et al., 1989).
Pregnancy is marked by a state of hypomagnesaemia. The serum magnesium concentration shows no gestational dependence until 33 weeks, at which point it continuously declines. Serum magnesium is not depressed further with the onset of labour at term. Patients in preterm labour have a significantly decreased serum magnesium concentration (Kurzel, 1991). This concentration was not dependent on whether the aetiology for the preterm labour was PROM, twin gestation, placental abruption, placenta praevia with bleeding or chorioamnionitis. The lower rate of the passage of Ca2+ and Mg2+ through preterm fetal membrane resulting in reduced availability of these cations in the maternal compartment is consistent with the data showing lower concentrations of these cations in maternal plasma in preterm labour than in term labour (Kiilholma et al., 1984
; Kidroni et al., 1989
). The data of the present study show lower permeability to Mg2+ of preterm fetal membrane than of term membrane. This would result in low amounts of Mg2+, as of Ca2+, reaching the myometrium and placenta in preterm labour compared with term labour. As a consequence, the myometrium should be more sensitive to activation by whichever physiological stimulant or signal it receives, and would tend to lead to preterm myometrial contractions. Furthermore, Mg2+ has recently been shown to enhance NOS activity (Pearson et al., 1998
). A reduction in Mg2+ would further result in diminished production of NO in the myometrium and more importantly in the placenta, and consequently a lower degree of quiescence of the myometrium in preterm cases.
Another important consequence of reduction in myometrium Mg2+ would be the effect on the magnesium-dependent ATPase in the sarcoplasmic reticulum (SR). This ATPase is responsible for the regulation of myoplasmic Ca2+ by transporting Ca2+ from the myoplasm to storage sites within the SR.
A reduction in SR-ATPase would result in higher Ca2+ ion concentration in the cytoplasm, which is also observed with the use of certain agents such as thapsigarigin, and would increase myometrial activity. Thus, a reduced paracrine effect of NO produced by placenta and inadequate removal of myoplasmic Ca2+ by SR in myometrial cells would synergistically result in increased myometrial contractility.
Recently, human fetal membranes were shown to release a calcium channel inhibitor and it was suggested that paracrine factors such as this inhibitor might contribute to relative quiescence of the preterm uterus (Emery et al., 1998). The release of this and many other secretory products could also depend on the availability of Ca2+ and Mg2+. It would be of interest to know whether the synthesis or secretion of this endogenous inhibitor in fetal membranes from preterm labour is reduced. Studies are underway in our laboratory to examine this interesting possibility.
In summary, the results of the present study showing markedly reduced permeability to Ca2+ and Mg2+ of fetal membranes from preterm labour suggest that this abnormality could be an important factor for the activation of the myometrium in preterm labour.
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Acknowledgments |
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Notes |
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References |
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Batra, S.C. and Popper, L.D. (1989) Characterisation of membrane calcium channels in nonpregnant and pregnant human uterus. Gynecol. Obstet. Invest., 27, 5761.[ISI][Medline]
Benech-Kieffer, F. (1995) Percutaneous absorption/penetration in vitro. Excised human skin standard protocol. 19. In Colipa, T.F. (ed.) Percutaneous Absorption/Penetration. Rue de la Loi 223/2.B-1040 Bruxelles.
Bennett, P.R., Chamberlain, G.V., Patel, L. et al. (1990) Mechanisms of parturition: the transfer of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid across fetal membranes. Am. J. Obstet. Gynecol., 162, 683687.[ISI][Medline]
Bronaugh, R.L., Stewart, R.F. and Simon, M. (1986) Methods for in vitro percutaneous absorption studies VII: use of excised human skin. J. Pharm. Sci., 75, 10941097.[ISI][Medline]
Buhimschi, I., Yallampalli, C., Dong, Y.-L. and Garfield, R.E. (1995) Involvement of a nitric oxide guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am. J. Obstet. Gynecol., 172, 15771584.[ISI][Medline]
Chaud, M.A., Franchi, A.M., de Astrada, M.B. et al. (1997) Role of nitric oxide on oxytocin-evoked contractions and prostaglandin synthesis in isolated pregnant rat uterus. Prostaglandins Leukotrienes Essential Fatty Acids, 57, 323329.[ISI][Medline]
Czylewska, K., Szary, B. and Waniewski, J. (1997) In vitro evaluation of peritoneal glucose transport in the rabbit. Med. Sci. Monit., 3, 637641.
Davidge, S.T., Baker, P.N., McLaughlin, M.K. and Roberts, J.M. (1995) Nitric oxide produced by endothelial cell increases production of eicosanoids through activation of prostaglandin H synthase. Circ. Res., 77, 274283.
Dennes, W.J.B., Slater, D.M. and Bennett, P.R. (1997) Nitric oxide synthase mRNA expression in human fetal membranes: a possible role in parturition. Biochem. Biophys. Res. Commun., 233, 276278.[ISI][Medline]
Emery, S.P., Idriss, E., Richmonds, C. et al. (1998) Human fetal membranes release a Ca++ channel inhibitor. Am. J. Obstet. Gynecol., 179, 989993.[ISI][Medline]
Gibb, W. (1998) The role of prostaglandins in human parturition. Ann. Med., 30, 235241.[ISI][Medline]
Godfrey, K. (1983) Compartmental Models and Their Application. Academic Press Ltd, London, New York.
Harrison, S.M., Barry, B.W. and Dugard, P.H. (1984) Effects of freezing on human skin permeability. J. Pharm. Pharmacol., 36, 261262.[ISI][Medline]
Johnson, P. (1993) Suppression of preterm labour: current concepts. Drugs, 45, 684692.[ISI][Medline]
Kidroni, G., Yagel, S., Menczel, J. et al. (1989) Studies of normal and prematurely ruptured human amniotic membranes: low calcium and magnesium in prematurely ruptured membranes. J. Perinat. Med., 17, 433438.[ISI][Medline]
Kiilholma, P.M., Gronroos, M., Erkkola, R. et al. (1984) The role of calcium, copper, iron and zinc in preterm delivery and premature rupture of fetal membranes. Gynecol. Obstet. Invest., 17, 194201.[ISI][Medline]
Knowles, R.G. and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J., 298, 249258.[ISI][Medline]
Kurzel, R.B. (1991) Serum magnesium levels in pregnancy and preterm labor. Am. J. Perinatol., 8, 119127.[ISI][Medline]
Lee, C.K., Uchida, T., Kitagawa, K. et al. (1994a) Relationship between lipophilicity and skin permeability of various drugs from an ethanol/water/lauric acid system. Biol. Pharmacol. Bull., 17, 14211424.
Lee, C.K., Uchida, T., Kitagawa, K. et al. (1994b) Skin permeability of various drugs with different lipophilicity. J. Pharm. Sci., 83, 562565.[ISI][Medline]
Malak, T.M. and Bell, S.C. (1994) Structural characteristics of term human fetal membranes: a novel zone of extreme morphological alteration within the rupture site. Br. J. Obstet. Gynaecol., 101, 375386.[ISI][Medline]
Malak, T.M., Mulholland, G. and Bell, S.C. (1994) Morphometric characteristics of the decidua, cytotrophoblast, and connective tissue of the prelabor ruptured fetal membranes. Ann. N. Y. Acad. Sci., 734, 430432.[ISI][Medline]
McLaren, J., Malak, T.M. and Bell, S.C. (1999) Structural characteristics of term human fetal membranes prior to labour: identification of an area of altered morphology overlying the cervix. Hum. Reprod., 14, 237241
Motta, A.B., Chaud, M.A. and de Gimeno, M.F. (1998) Regulation by nitric oxide of prostaglandin E synthesis and spontaneous motility in rat uterine tissue. Prostaglandins Leukotrienes Essential Fatty Acids, 58, 333338.[ISI][Medline]
Nanno, H., Sagawa, N., Itoh, H. et al. (1998) Nitric oxide metabolite concentrations in maternal plasma decrease during parturition: possible transient down-regulation of nitric oxide synthesis. Mol. Hum. Reprod., 4, 609616.[Abstract]
Pearson, P.J., Evora, P.R.B., Seccombe, J.F. et al. (1998) Hypomagnesemia inhibits nitric oxide release from coronary endothelium: protective role of magnesium infusion after cardiac operations. Ann. Thorac. Surg., 65, 967972.
Philippe, M. (1998) Cellular mechanisms underlying magnesium sulfate inhibition of phasic myometrial contractions. Biochem. Biophys. Res. Commun., 252, 502507.[ISI][Medline]
Popper, L.D., Batra, S. and Akerlund, M. (1989) The effect of magnesium on calcium uptake and contractility in the human myometrium. Gynecol. Obstet. Invest., 28, 7881.[ISI][Medline]
Rasmay, B., Sooranna, S.R. and Johnson, M.R. (1996) Nitric oxide synthase activities in human myometrium and villous trophoblast throughout pregnancy. Obstet. Gynecol., 87, 249253.
Sahin-Tóth, M., Kukor, Z. and Tóth, M. (1997) Tetrahydrobiopterin preferentially stimulates activity and promotes subunit aggregation of membrane-bound calcium-dependent nitric oxide synthase in human placenta. Mol. Hum. Reprod., 3, 293298.[Abstract]
Salvemini, D., Settle, S.L., Masferrer, J.L. et al. (1995) Regulation of prostaglandin production by nitric oxide: an in vivo analysis. Br. J. Pharmacol., 114, 11711178.[Abstract]
Seeds, A.E. (1970) Osmosis across term human placental membranes. Am. J. Physiol., 219, 551554.[Medline]
Sladek, S.M., Magness, R.R. and Conrad, K.P. (1997) Nitric oxide and pregnancy. Am. J. Physiol. [Regulatory Integrative and Comparative Physiology], 41, R441R463.
Syal, A., Okawa, T., Vedernikov, Y. et al. (1999) Effects of placental tissue on inhibition of uterine contraction by nitric oxide donors. Am. J. Obstet. Gynecol., 181, 415418.[ISI][Medline]
Yallampalli, C. and Garfield, R.E. (1993) Role of nitric oxide in the uterus during pregnancy and parturition. Curr. Top. Pharmacol., 2, 6990.
Yallampalli, C., Buhimschi, I., Chwallisz, K. et al. (1996) Preterm birth in rats produced by synergistic action of nitric oxide inhibitor (NG-nitro-L-arginine methyl ester) and an antiprogestin (onapristone). Am. J. Obstet. Gynecol., 175, 207212.[ISI][Medline]
Submitted on February 15, 2000; accepted on May 30, 2000.