Spontaneous Human Myometrial Contractility in the Third Trimester of Pregnancy in Relation to Past Mode of Delivery.

OBJECTIVE: It is well established that women with a previous vaginal delivery have higher success rates in relation to vaginal birth after cesarean than those without. The aim of this study was to examine the effect of past mode of delivery on contractile parameters of human myometrium in vitro. STUDY DESIGN: Myometrial strips were excised from 64 women at cesarean delivery (CD) and recordings of spontaneous contractile activity analyzed and compared across three clinical groups: (1) women with no previous delivery (Group 1); (2) women with CD only (Group 2); and (3) women with a history of vaginal delivery and CD (Group 3). RESULTS: Myometrial samples from women in Group 3, women who had a previous vaginal delivery, had a significantly greater maximum amplitude of contractions (p < 0.05), a greater force (mean contractile force) of contractions (p < 0.01), and a faster rate of rise (p < 0.01) and relaxation of contractions (p < 0.05) than those in Groups 1 and 2. CONCLUSION: Many of the functional parameters of human uterine contractions are altered, or enhanced, in the women who have had a previous vaginal delivery, when compared with those without. This may partly explain the clinical differences observed in labor.

Effects of maternal parity on response of human myometrium to oxytocin and ergometrine in vitro.

OBJECTIVE: The aim of this study was to compare the effects of oxytocin and ergometrine on the intrinsic contractile parameters of human uterine smooth muscle at term between primiparous and multiparous women. STUDY DESIGN: Myometrial biopsies were obtained from women undergoing planned caesarean section at term. The biopsies were dissected into eight uniform strips and mounted in tissue baths for isometric recording. The strips were challenged with increasing concentrations of oxytocin and ergometrine. Parameters of contractile activity, including mean contractile force (MCF) and maximum amplitude of contractions (MAMP) were recorded and analysed. Results were compared between primiparous (Group 1) and multiparous (Group 2) women. RESULTS: Myometrial biopsies were obtained from n = 11 donors (88 tissue strips), of which n = 5 were Group 1 and n = 6 were Group 2. In relation to oxytocin, the MAMP value observed was significantly greater in Group 2 than in Group 1 (151 ±â€¯18mN vs 67 ±â€¯14mN, P < 0.01). Regarding ergometrine, the MCF response was greater in Group 2 samples (24 ±â€¯10 mN) than that in Group 1 (18 ±â€¯2mN) (P < 0.05). CONCLUSION: Our findings highlight that women in a first pregnancy have a decreased response to both oxytocin and ergometrine in an in vitro setting when compared with women in a subsequent pregnancy, and this may have clinical implications regarding the management of postpartum haemorrhage in this cohort.

Synthesis of α-methylstilbenes using an aqueous Wittig methodology and application toward the development of potent human aromatase inhibitors.

The development of aqueous Wittig methodology for the synthesis of α-methylstilbenes using tripropylphosphine-derived phosphonium salts is described. The Wittig olefination reaction was high yielding and allowed isolation of stilbenes by simple filtration and washing with water. The novel phosphonium salts employed were accessed via a highly efficient, regioselective addition of hydrogen bromide to styrenes. Application of the α-methylstilbenes toward the synthesis of a collection of stilbenoid-triazoles is reported and their inhibition of CYP450 19A1 (aromatase) investigated. The overall structure-activity profile provided additional evidence on the aryl halide-ketone bioisostere hypothesis and identified 6c as a potent inhibitor of aromatase in vitro (Ki = 8 nM).

Maternal parity and functional contractility of human myometrium in vitro in the third trimester of pregnancy.

OBJECTIVE: It is well established that the duration of the first and second stages of labor are shorter in parous women than in their nulliparous counterparts, a phenomenon not well understood. The aim was to examine the effect of maternal parity on contractile parameters of human myometrium. DESIGN: Myometrial strips were excised from n = 74 women at cesarean delivery and recordings of contractile activity analyzed and compared across three clinical groups: 1. No previous delivery (P0); 2. One previous delivery (P1); 3. Greater than one previous delivery (P>1). RESULTS: There was a trend towards greater mean contractile force in the P>1 group than the P1 and P0 groups (P = 0.412). Frequency of contractions was less in the P1 group than in the P0 and P>1 groups(P = 0.027). No differences were observed in relation to all other parameters. CONCLUSION: Excluding frequency, no intrinsic differences were observed in the functional parameters of human uterine contractions in relation to parity.

Effects of the KIR7.1 Blocker VU590 on Spontaneous and Agonist-Induced Contractions of Human Pregnant Myometrium.

KIR7.1, an inwardly rectifying K+ channel, plays a critical role in regulating uterine excitability during pregnancy and has been suggested as a potential new target for the treatment of conditions arising from dysfunctional uterine contractility, for example, atonic postpartum hemorrhage. The aim of this study was to investigate the effects of the selective KIR7.1 blocker, VU590, on both spontaneous and agonist-stimulated contractions of human pregnant myometrium in vitro. At a concentration of 20 µmol/L, VU590 significantly increased the mean contractile force and the frequency of spontaneous contractions ( P < 0.05) when compared to vehicle-treated tissues. However, there was a significant ( P < 0.0001) monoexponential decay in amplitude with time of exposure. When VU590 was coadministered with EC50 concentration of the uterotonics oxytocin, ergometrine, or carboprost, the only significant changes were an immediate decrease in the amplitude of oxytocin- and carboprost-induced contractions and a delayed reduction in amplitude and an increase in the frequency of ergometrine-induced contractions. Amplitude to all 3 agents in the presence of VU590 showed a monoexponential decay with time of exposure ( P < 0.0001). We conclude that VU590 modifies the contractility of pregnant human myometrium in support of a role for KIR7.1 in regulating that process. However, VU590 in vitro does not produce the types of contraction, either alone or in combination with other uterine stimulants that would suggest its usefulness as a first- or second-line clinical uterotonic agent.

In vitro contractile effects of agents used in the clinical management of postpartum haemorrhage.

Uterine atony is a major cause of postpartum haemorrhage and maternal mortality. However, the comparative pharmacology of agents used to treat this condition is poorly understood. This study evaluates, using human pregnant myometrium in vitro, a range of contractile parameters for agents used in the clinical treatment of atonic postpartum haemorrhage. The effects of oxytocin, carbetocin, ergometrine, carboprost, syntometrine and misoprostol were investigated in 146 myometrial strips from 19 donors. The potency and maximal response values were obtained, and compared, using both maximal amplitude and mean contractile force as indices of contraction. Single, EC50 concentrations of the agents were administered and both force and contraction peak parameters were compared during a 15-min exposure. Differences were considered significant when P<0.05. There were no significant differences in the peak amplitude of response between agents, except for misoprostol, which was inactive. There was a wide difference in potencies using both measures of contractility, with oxytocin and carbetocin being the most potent. The most important difference between the agents was in their ability to increase the mean contractile force, with oxytocin superior to all agents except syntometrine. In single dose experiments, mean contractile force was the parameter that separated the agents. In this respect, oxytocin was not statistically different from carboprost or syntometrine, but was superior to all other agents. These findings support a clear role for oxytocin as the first line agent for treatment of postpartum haemorrhage and raise doubts about the potential clinical usefulness of misoprostol.

Maternal Age and Contractility of Human Myometrium in Pregnancy.

There is controversy as to whether maternal age exerts an influence on the contractility of human myometrium in pregnancy. The aim of this study was to examine a series of functional contractile parameters of human myometrium in vitro, over a broad range of maternal ages. Myometrial tissue specimens were obtained at cesarean delivery from 32 women with maternal ages ranging from 28 to 52 years. Using in vitro recordings, a number of contractile parameters including maximal amplitude, mean contractile force, time to maximal amplitude, maximum rate of rise, and occurrence of simple and complex (biphasic and multiphasic) contractions were examined for spontaneous and induced contractile activity. The relationship between maternal age and individual parameters was evaluated using linear regression analysis. For all contractile parameters examined, for both spontaneous and induced contractions, no significant correlation was observed with maternal age between 28 and 52 years. The mean maximum amplitude values for spontaneous and oxytocin-induced contractions were 23 ± 3 and 43 ± 5 mN, respectively. The mean contractile forces for spontaneous and oxytocin-induced contractions were 1.5 ± 0.2 and 6.5 ± 0.9 mN, respectively. There was no variation in the proportion of biphasic or multiphasic contractions with maternal age. These results indicate there is no significant functional impairment of uterine contractility and no lack in responsiveness of myometrium in vitro, in the older mother. These findings do not support the concept that there may be a biological basis for dysfunctional labor or increased cesarean delivery rates in older parturients.

Human uterine lower segment myometrial cell and nuclear volume at term: influence of maternal age.

Little is known about the cytoarchitecture of human myometrial cells in pregnancy, and whether or not this may be influenced by maternal characteristics such as age, parity and body mass index (BMI). The aim of this study was primarily to evaluate human myometrial smooth muscle cell (SMC) and nuclear volume in the third trimester of human pregnancy, and secondarily to investigate if these parameters are altered in relation to the maternal characteristics outlined above. Myometrial biopsies were obtained from 30 women undergoing elective caesarean delivery at term. One-micrometer sections were prepared for light microscopy and 100-nm sections for electron microscopy. The nucleator technique was used to assess nuclear volume from the light microscopy images. Point-counting methodology was used on transmission electron micrographs to assess the percentage of the cell volume occupied by the nucleus. Cell volume was calculated from these measurements. The euchromatin to heterochromatin (Eu/Het) ratio was determined to ascertain whether differences in nuclear volume were due to an increased range of genes being transcribed. The mean (±â€…SEM) nuclear volume was 175 ±â€…10 µm(3) , the nucleus occupied 1.5 ±â€…0.1% of the SMC and the mean cell size was 14 047 ±â€…1352 µm(3) . The Eu/Het ratio was 7.54 ±â€…0.4. The mean volume of heterochromatin and euchromatin in the nucleus was 21.5 ±â€…1.7 and 149 ±â€…9 µm(3) , respectively. A multivariate regression analysis revealed that advanced maternal age was associated with an increase in the percentage of the cell occupied by nucleus (R(2)  = 0.32, P = 0.004). There were no other significant effects of maternal age, BMI or parity on the measured parameters. These findings provide reliable volumes for human myometrial cells and their nuclei at term gestation, and show that nuclear volume fraction may be influenced by maternal age.

The effects of methyl palmitate, a putative regulator from perivascular fat, on the contractility of pregnant human myometrium.

AIMS: Methyl palmitate is thought to cause relaxation in vascular smooth muscle by opening voltage-activated potassium channels. We have tested the hypothesis that methyl palmitate, a putative regulator from perivascular fat, is an inhibitor of the contractility of human pregnant myometrium and that its effects might partially explain the higher incidence of dysfunctional labor in obese women compared to those with normal body mass indices. MAIN METHODS: Strips of myometrium obtained with informed consent from women undergoing elective cesarean section at term were mounted in organ baths. Strips stimulated with oxytocin (1nM) or KCl (30mM) were exposed to cumulatively increasing concentrations of methyl palmitate up to 10µM. Similar strips were exposed to cumulative addition of the potassium channel blockers 4-aminopyridine and tetraethylammonium. The contractility of the strips was monitored and analyzed using conventional methods. KEY FINDINGS: Methyl palmitate failed to inhibit oxytocin- or KCl-induced contractions over the concentration range tested. In fact, it exerted a slight excitatory effect in the presence of KCl, though not in the presence of oxytocin. The contractility of naïve strips was unaltered by exposure to 1µM methyl palmitate. Both 4-aminopyridine and tetraethylammonium produced concentration-dependent contractions of human pregnant myometrium providing pharmacological evidence for the presence of voltage-activated potassium channels in this preparation. SIGNIFICANCE: Our findings do not support the hypothesis that methyl palmitate is an inhibitor of human pregnant myometrial contractility. Alternate hypotheses must be pursued to explain the higher incidence of dysfunctional labor in obese women.

Discovery of a new class of cinnamyl-triazole as potent and selective inhibitors of aromatase (cytochrome P450 19A1).

Synthesis of a novel class of natural product inspired cinnamyl-containing 1,4,5-triazole and the potent inhibition of human aromatase (CYP 450 19A1) by select members is described. Structure-activity data generated provides insights into the requirements for potency particularly the inclusion of an aryl bromide or chloride residue as a keto-bioisostere.

The influence of smooth muscle content and orientation in dissected human pregnant myometrial strips on contractility measurements.

This study examined the hypothesis that the force generated by myometrial strips from pregnant women is influenced by the smooth muscle content and fibre orientation of the strips and that correcting for these structural variables will provide a more accurate measure of contractility. Myometrial strips (n=72) were contracted by exposure to KCl, oxytocin, U44619 and phenylephrine and maximum responses were recorded. Morphological techniques were used to determine the cross-sectional area of the strips, the area occupied by smooth muscle and the area occupied by smooth muscle longitudinal in the strip. Maximum responses to contractile agents were expressed in terms of these three variables. The mean cross sectional area of strips was 2.01 ± 0.06 mm(2), of which 50% was smooth muscle, and 18% was smooth muscle longitudinal in the strip (n=72). There was much heterogeneity in responses, smooth muscle content and fibre orientation. Correction for morphological variability did not improve the heterogeneity in responses where coefficients of variation among strips from the same donor ranged from 43% to 63% when expressed in relation to longitudinal smooth muscle cross-sectional area. The standard method of preparation of myometrial strips for in vitro recording results in samples that are not structurally uniform. Correcting for the known structural variables does not provide a more accurate measure of maximum contractile responses. Because of the heterogeneity shown here, experiments that are dependent upon accurate estimation of maximum contractile responses require a large number of replicates to reach meaningful conclusions.

Investigation of aryl halides as ketone bioisosteres: refinement of potent and selective inhibitors of human cytochrome P450 19A1 (aromatase).

Bioisosteric replacement of cyclic ketone functionality with aryl halides was investigated on a centrally-flexible, five-component 1,2,3-triazole-containing pharmacophore, resulting in enhanced inhibition of aromatase (CYP450 19A1). Structure-activity data generated from both syn- and anti-aldol precursors provides significant insights into the requirements for enhanced potency, validating this novel ketone-to-aryl halide bioisostere hypothesis.

The effects of extracellular calcium-sensing receptor ligands on the contractility of pregnant human myometrium in vitro.

Ligands for extracellular calcium-sensing (CaS) receptors inhibit oxytocin-induced contractions of the rat's uterus. In this study, we investigated whether the CaS receptor ligands calindol, cinacalcet, and calhex 231 have similar effects on pregnant human myometrium. We compared their effects to those of the calcium-channel blocker nifedipine. In conventional concentration-effect experiments, both the mean contractile force (MCF) and the maximum amplitude of contractions induced by 1 nmol/L oxytocin were inhibited by nifedipine. Calindol and cinacalcet were ineffective as inhibitors, while calhex-231 produced partial inhibition. When single 10 µmol/L doses were applied calhex-231 produced a slowly developing inhibition, reducing the MCF to 38%, and amplitude to 34%, of vehicle controls after 1 hour. In similar experiments, calindol was ineffective while cinacalcet weakly inhibited only the amplitude. Immunohistochemistry revealed sparse expression of CaS receptors in pregnant human myometrium.

Stereology of human myometrium in pregnancy: influence of maternal body mass index and age.

OBJECTIVE: Knowledge of the stereology of human myometrium in pregnancy is limited. Uterine contractile performance may be altered in association with maternal obesity and advanced maternal age. The aim of this study was to investigate the stereology of human myometrium in pregnancy, and to evaluate a potential influence of maternal body mass index (BMI) and age. STUDY DESIGN: Biopsies of human myometrium were obtained from 57 women at cesarean section (n = 26, n = 13, n = 18 normal, overweight and obese BMI, respectively), and volume fractions of smooth muscle and extracellular matrix were assessed using stereologic techniques. RESULTS: The smooth muscle constituted 65.2% ± 8.9% (standard deviation) and the extracellular matrix 32.6% ± 7.7% (standard deviation) (n = 57). There was no correlation observed between maternal BMI, age, or parity with the fractional volumes of either smooth muscle or extracellular matrix. CONCLUSION: These results outline the stereology of human myometrium in pregnancy. Putative functional differences in contractility, pertaining to obese or older mothers, are not related to smooth muscle content.

Immunohistochemical and functional studies on calcium-sensing receptors in rat uterine smooth muscle.

1. Activation of calcium-sensing receptors (CaS) leads to relaxation of vascular smooth muscle. However, the role of CaS in uterine smooth muscle is unknown. Therefore the aim of the present study was to investigate the expression and function of CaS in the uterus. 2. The expression of CaS in the oestrogen-dominated rat uterus was investigated using immunohistochemistry. The effects of putative CaS ligands on oxytocin-induced contractions of longitudinally orientated uterine strips from oestrogen-dominated rats were determined at reduced extracellular Ca²âº concentrations using conventional organ bath techniques. 3. Immunohistochemical evidence showed the presence of CaS in the endometrium and smooth muscle layers of the rat uterus. Oxytocin-induced contractions were inhibited by cations (Gd³âº > Ca²âº = Mg²âº), polyamines (spermine > spermidine) and the positive allosteric modulators cinacalcet and calindol. However (R)- and (S)-cinacalcet were equipotent, indicating a lack of stereoselectivity, and the negative allosteric modulator calhex-231 also caused dose-dependent relaxation. In addition, although intermediate-conductance calcium-activated potassium channels and cytochrome P450-dependent signal transduction have been implicated in CaS-induced relaxation of vascular smooth muscle, neither Tram-34 nor miconazole (1 µmol/L), which block these pathways, respectively, had any effect on the ability of cinacalcet to inhibit oxytocin-induced contractions. 4. Calcium-sensing receptors are expressed in smooth muscle layers of the rat uterus and their ligands produce potent relaxation of longitudinally orientated uterine strips. However, the pharmacological profile of inhibition of contractility by CaS ligands is not consistent with a role for CaS in the regulation of uterine contractility in the rat.

Discovery of a novel class of aldol-derived 1,2,3-triazoles: potent and selective inhibitors of human cytochrome P450 19A1 (aromatase).

The discovery of a novel five-component 1,2,3-triazole-containing pharmacophore that exhibits potent and selective inhibition of aromatase (CYP 450 19A1) is described. All compounds are derived from an initial aldol reaction of a phenylacetate derivative with an aromatic aldehyde. Structure-activity data generated from both syn- and anti-aldol adducts provides initial insights into the requirements for both potency and selectivity.

Methodology and pharmacological analysis of effects of uterotonic compounds in human myometrium in vitro.

OBJECTIVE: The methodology used to evaluate contractile effects of uterotonic agents in human myometrium in vitro varies. The are no studies evaluating the reliability of these commonly used techniques. STUDY DESIGN: Myometrial strips (n=72) were exposed to 3 known uterotonic agents: oxytocin, U46619, and phenylephrine. The negative log of the molar concentration of the agonist that produces a half-maximal response (pEC50) and maximal response values were obtained, and compared, when either amplitude or mean force was used as indices of contraction. All data were expressed as a percentage of KCl elicited actvity. RESULTS: Using pEC50 measurements, the order of potency was oxytocin greater than U46619 greater than phenylephrine for both indices, whereas the order of maximal response varied between mean force and amplitude. The coefficient of variation was lowest for pEC50 measurements, highest for maximal force estimations, and overall was 10-48% between, and 2-27% within, donor samples. CONCLUSION: These findings support the use of pEC50 measurements for in vitro experiments using uterotonic agents and outline the variability that occurs for such myometrial experiments.

The effects of commercial preparations of herbal supplements commonly used by women on the biotransformation of fluorogenic substrates by human cytochromes P450.

The study set out to determine the potential for commercially available preparations of black cohosh (Actaea racemosa), chaste tree berry (Vitex agnus-castus), crampbark (Viburnum opulus) and false unicorn (Chamaelirium luteum) to inhibit the major human drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 as well as CYP1A1 which activates some carcinogens. In vitro microplate-based assays using cDNA-expressed CYP450 isoforms and fluorogenic substrates were used. Components of the commercial herbal preparations interfered with the assays and limited the concentration ranges that could be tested. Nevertheless, the fluorogenic assays were robust, reproducible and easy to perform and thus are still useful for initial screening for potential herb-drug interactions. None of the preparations affected CYPs 1A1 or 2C9 at the concentrations tested but all preparations inhibited some of the enzymes with potencies around 1 µg/mL. The three most potent interactions were: chaste tree berry and CYP2C19 (IC50) 0.22 µg/mL); chaste tree berry and CYP3A4 (IC50) 0.3 µg/mL); black cohosh and CYP2C19 (IC50) 0.37 µg/mL,). Thus, the study successfully identified the potential for the commercial herbal preparations to inhibit human drug metabolizing enzymes. Whether this potential translates into clinically significant herb-drug interactions can only be confirmed by appropriate in vivo studies.

Effects in rats of maternal exposure to raspberry leaf and its constituents on the activity of cytochrome p450 enzymes in the offspring.

The goal of our study was to determine whether maternal exposure to red raspberry leaf (RRL) and its constituents can permanently alter biotransformation of fluorogenic substrates by cytochrome P450 (CYP) in the livers of male and female offspring. Nulliparous female rats received vehicle, raspberry leaf, kaempferol, quercetin, or ellagic acid orally once breeding had been confirmed until parturition. Hepatic microsomes were prepared from animals at birth (postnatal day 1 [PND1]), weaning (PND21), PND65, and PND120 to determine the biotransformation of 8 fluorogenic substrates. The pattern of biotransformation of all but 2 of the substrates was gender specific. Maternal consumption of RRL increased biotransformation of 3 substrates by female offspring at PND120 resulting in a more masculine profile. Kaempferol and quercetin had a similar effect to RRL. These results suggest that maternal consumption of either RRL or some of its constituents leads to long-term alterations of CYP activity in female offspring.

Human cytochrome P450 liability studies of trans-dihydronarciclasine: a readily available, potent, and selective cancer cell growth inhibitor.

The cytochrome P45O activities of the naturally occurring Amaryllidaceae alkaloid narciclasine (3), isolated from Narcissus pseudonarcissus, and synthetic derivative trans-dihydronarciclasine (5) are reported. While narciclasine was found to possess potent inhibitory activity to human CYP3A4, its dihydro analogue was inactive. This study revealed that the C1-C10b double bond is required for inhibition of this crucial metabolizing enzyme. Compound 5 also demonstrated no inhibition of the related human cytochromes CYP19 and CYP1A1. This study elevates the status of trans-dihydronarciclasine (5) as a highly privileged, readily available molecule, with potent and selective anticancer activity.