M.G. Elder

Preterm labor: Stimulation of arachidonic acid metabolism in human amnion cells by bacterial products

There is a strong association between preterm labor and infection. Some potentially pathogenic bacteria have phospholipase activity, and it has been suggested that release of phospholipase from these organisms may increase prostaglandin E2 synthesis in amnion cells and hence initiate preterm labor. In this study we established monolayer amnion cell cultures from tissue collected at elective cesarean section at term before labor. Cells were prelabeled with tritiated arachidonic acid and then further incubated after addition of 2%, 5%, or 10% (vol/vol) filtered medium in which either group B beta-hemolytic streptococcus, Streptococcus viridans, Escherichia coli, Bacteroides fragilis, or Lactobacillus had been growing. Tritiated arachidonic acid and its metabolites released by the amnion cells in these or control incubates were extracted from culture medium and separated by high-performance liquid chromatography. Addition of conditioned medium from each of the organisms with the exception of Lactobacillus caused an increase in overall arachidonic acid metabolism. There was an increase in the ratio of cyclooxygenase to lipoxgenase metabolism and in prostaglandin E2 production in particular when compared to controls. The profile of arachidonic acid metabolism in amnion cells following addition of filtered bacterial medium resembled that obtained from amnion cells cultured following spontaneous labor. We suggest that abnormal bacterial colonization of the genital tract may lead to an increase in arachidonic acid metabolism in amnion cells with an increase in prostaglandin E2 production and the consequent initiation of preterm labor.

Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulatory role at the feto-maternal interface.

Feto-placental vessels lack innervation, hence control of this circulation is dependent on locally produced and circulating vasoactive factors. Functional studies have presented evidence that nitric oxide, a potent vasodilator and platelet anti-aggregating agent, may be generated into the feto-placental circulation, contributing to control of vascular tone. In view of the absence of nerves supplying the placenta the source of NO is likely to be endothelial. We have therefore investigated the localization of endothelial constitutive nitric oxide synthase (ecNOS) in human normal full-term placentae, using immunocytochemistry, with rabbit antiserum to a synthetic peptide, corresponding to amino acid residues 1172-1186 of human and bovine ecNOS. On Western blots of partially purified NO synthase extracted from placenta, the peptide antiserum reacted exclusively with a single protein band of approximately 135kDA. Immunoreactivity in tissue sections was localized to endothelium of umbilical artery and vein, and appeared uniform in sections at different levels along the cord. Staining in chorionic vessels was much more variable; it was present mainly in the larger vessels close to the cord where it had a patchy distribution. Staining was not seen in the endothelium of small feto-placental vessels. Strong immunoreactivity was evident in the syncytiotrophoblast of the placenta, although the intensity of staining was variable, being weaker along stem villi and strongest along terminal villi. The differential distribution and intensity of nitric oxide synthase immunoreactivity in the human placenta might indicate that locally produced, and in particular trophoblast-derived nitric oxide may play a pivotal role both in control of feto-placental vascular tone and as a platelet anti-aggregating agent in the utero-placental circulation.

Effect of bacterial products on prostaglandin E production by amnion cells.

An in-vitro model was set up to study a possible causal relation between bacterial colonisation of extraplacental membranes and spontaneous preterm labour. When amnion cells in tissue culture were exposed to bacterial products, their prostaglandin E output rose considerably. The degree of response varied with the organism. These findings support the theory that bacterial products may be responsible for the premature onset of labour.

Spontaneous early preterm labour associated with abnormal genital bacterial colonization

Summary. The association between infection and preterm labour was studied in 72 women in spontaneous preterm labour between 26 and 34 weeks gestation and in 26 control subjects having an elective caesarean section at the same gestational age. The genital microbial flora of each group was studied comprehensively and included mycoplasmas, chlamydiae, ureaplasmas and anaerobes. Subsequent neonatal infection and chorioamnionitis was also studied. Abnormal bacterial colonization, the presence of ureaplasmas, heavy growth of mycoplasmas and chorioamnionitis were all found significantly more often in the study group. This supports the premise that a significant proportion of idiopathic preterm labour is associated with infection and this may permit better prediction and prevention of preterm birth. The continued use of tocolytics should depend upon the identification of the presence or absence of infection. Infection appeared to be the result rather than the cause of ruptured membranes. A recommendation with respect to the classification of abnormal or normal bacterial colonization between 26 and 34 weeks is suggested on the basis of strict criteria.

The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility.

The effects of the lipoxygenase products of arachidonic acid, 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B4 (LTB4), on the spontaneous contractility of lower uterine segment human myometrial strips obtained prior to labour have been studied in vitro. 5-HETE gave a dose- dependent (10-500ng) increase in both the rate of contractions and overall contractility of myometrial strips while 12-HETE and LTB4 had no effect at the same concentrations. Prostaglandin F2 alpha (50ng) contracted all myometrial strips in a similar pattern to 5-HETE but was approximately 10 times more potent. The effect of 5-HETE may be direct or perhaps indirect via interaction with the cyclo-oxygenase pathway. The findings do not disprove the contention that the onset of parturition may be characterised by a switch in arachidonic acid metabolism in intra-uterine tissues from lipoxygenase to cyclo-oxygenase products.

Prostaglandin production and stimulation by angiotensin II in the isolated perfused human placental cotyledon

Levels of prostaglandins E and F2 alpha, thromboxane B2, and 6-oxo-prostaglandin F1 alpha were measured by radioimmunoassay in the maternal and fetal effluents of isolated human placental cotyledons perfused in vitro. All prostaglandins measured were released in greater amounts by the maternal side than by the fetal side of the perfused cotyledon although there were no consistent concentration gradients between the two sides. The approximate rank order of prostaglandin release into the maternal side was thromboxane B2 greater than prostaglandin F2 alpha congruent to prostaglandin E congruent to 6-oxo-prostaglandin F1 alpha, and that into the fetal side was thromboxane B2 congruent to prostaglandin F2 alpha congruent to prostaglandin E congruent to 6-oxo-prostaglandin F1 alpha. Injection of angiotensin II (0.5 microgram) into the fetal circulation stimulated prostaglandin E and 6-oxo-prostaglandin F1 alpha but not thromboxane B2 and prostaglandin F2 alpha release into the fetal circulation and had no effect on maternal release. Angiotensin II (0.5 microgram) had no effect on either side of the perfused cotyledon when injected into the maternal circulation. It is proposed that prostaglandin release into both maternal and fetal circulations may be flow-dependent and that the angiotensin II-stimulated release of prostaglandin E and 6-oxo-prostaglandin F1 alpha may serve to modulate the vasoactive actions of angiotensin II on the fetal vasculature.

Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies.

1 It has been suggested that a deficiency of nitric oxide (NO) may explain many of the pathophysiological features of pre‐eclampsia (PE) and intra‐uterine (foetal) growth retardation (IUGR). To elucidate further the role of NO in the pathophysiology of pregnancy we have determined the relative amount and activity of NO synthase (NOS) in first trimester and normal‐term placental tissues, as well as in the placenta and umbilical cord in pregnancies complicated by PE and IUGR, using NG‐nitro‐L‐[2,3,4,5‐3H]‐arginine ([3H]‐L‐NOARG) binding, quantitative in vitro autoradiography, [3H]‐arginine to [3H]‐citrulline conversion and Western blotting. 2 Specific, high affinity (KD = 38 nM) [3H]‐L‐NOARG binding was demonstrated in the villous trophoblast of normal‐term placentae. Binding was calcium‐independent, stereoselective and exhibited a rank order of inhibition by NOS inhibitors and substrate (L‐NOARG ≥ L‐NMMA ≥ 7‐NI > L‐NAME > L‐Arg ≥ L‐NIO > ADMA). 3 [3H]‐L‐NOARG binding density and NOS activity were both significantly greater in placental tissues from first trimester and PE or IUGR complicated pregnancies compared to normal‐term placentae. 4 Western blotting, using an endothelial NOS peptide antiserum, demonstrated a ∼ 140 KDa protein band in placental extracts and indicated that the amount of immunoreactive material was significantly greater in first trimester compared to normal‐term placentae. 5 Specific [3H]‐L‐NOARG binding was also localized to the endothelial lining of umbilical arteries and veins, binding density being greater in the artery than the vein. [3H]‐L‐NOARG binding to the umbilical artery endothelium was significantly lower in PE and IUGR complicated pregnancies compared to normal‐term controls. 6 The role of trophoblast‐derived NO in human placental pathophysiology remains to be established, but differences in the amount of placental [3H]‐L‐NOARG binding, NOS activity and immunoreactive material indicate that expression of NOS in the villous trophoblast falls during pregnancy. Conversely, the apparent reduction in NOS in the umbilical artery endothelium in PE and IUGR complicated pregnancies may be indicative of endothelial dysfunction.

Evidence of chlamydial infection in infertile women with and without fallopian tube obstruction

This study investigates the prevalence of Chlamydia trachomatis antibodies in 164 infertile women who underwent diagnostic laparoscopy and dye insufflation as part of routine infertility investigations. C. trachomatis antibodies were found in 36 (22%) of 164 infertile women, which was significantly more than the prevalence of antibodies in a control group (22 of 200, 11%). C. trachomatis antibodies were found in 25 (35.7%) of 70 infertile women who had laparoscopically verified peripheral tubal disease. This was significantly more than the prevalence of C. trachomatis antibodies in infertile women with normal fallopian tubes (6 of 52, 11.5%). The prevalence of C. trachomatis antibodies in infertile patients with laparoscopically verified cornual disease was similar to those without cornual disease. C. trachomatis was not isolated from any of the patients studied. This study confirms that past chlamydial salpingitis is associated with the development of peripheral fallopian tube obstruction with resultant infertility.

The actions of prostaglandins and their interactions with angiotensin II in the isolated perfused human placental cotyledon

Summary. The prostaglandins PGE1, PGE 2, PGD 2, PGF 2α., U46619 and 6β‐PGIl were administered as bolus injections both separately and in combination with angiotensin II into the fetal circulation of isolated human placental cotyledons perfused in vitro. PGF2α, and PGD2, caused small dosedependent increases in fetal perfusion pressure when compared with U46619 which acted as an extremely potent vasoconstrictor of the fetal‐placental vasculature. PGE1, caused very small dose‐dependent decreases in fetal perfusion pressure when injected on its own. In combination with angiotensin 11, PGE1, PGD2, and 6β‐PG11, caused significant, dose‐related attenuations of the angiotensin II vasoconstrictive response whereas PGE2, PGF2α, and U46619 potentiated the response. Injections of angiotensin II after the infusion of indomethacin into the fetal circulation resulted in a potentiation of angiotensin II induced vasoconstriction. The results indicate that prostaglandins exert their effects on the fetal‐placental circulation by modulating the actions of angiotensin II.

The effects of the components of the renin-angiotensin system on the isolated perfused human placental cotyledon

Angiotensins I, II, and III, renin substrate, and des-Asp1-angiotensin I were injected as a bolus into either the maternal or fetal circulation of human placental cotyledons perfused in vitro. All drugs tested produced dose-related increments in fetal perfusion pressure when injected into the fetal circulation, with the order of potency being angiotensin I approximately equal to angiotensin II approximately equal to angiotensin III greater than or equal to des-Asp1-angiotensin I greater than or equal to renin substrate. The responses to all the drugs could be blocked by the competitive inhibitor of angiotensin II, (Sar1, Ala8)-angiotensin II, but only the actions of angiotensin I, renin substrate, and des-Asp1-angiotensin I could be blocked by angiotensin converting enzyme inhibitor. When the agents were injected into the maternal circulation, only angiotensins II and III caused dose-related increments in fetal perfusion pressure. Possibly, the placenta may be the main site of conversion of angiotensin I to angiotensin II in the fetoplacental unit, and angiotensin II produced by the placenta could act locally to control fetoplacental blood flow.