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 (pEC 50 ) 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 pEC 50 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 pEC 50 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 pEC 50 measurements for in vitro experiments using uterotonic agents and outline the variability that occurs for such myometrial experiments.
Research has focused on factors modulating human uterine contractility during pregnancy, and labor is important in terms of understanding the normal physiology of such events and also for evaluating novel therapeutic interventions for pathophysiological processes such as preterm labor, dysfunctional uterine activity, and postpartum hemorrhage. These clinical problems are associated with significant perinatal, and maternal morbidity and mortality, in clinical practice. The use of in vitro pharmacological techniques for investigating human uterine contractility has become more widespread in recent years, and, unlike previously used clinical tocographic technology, the in vitro approach allows for objective assessment of contractile performance and greater understanding of the potential effects of inhibitory and uterotonic compounds.
In addition, technological advances in the design of modern data acquisition systems, and their associated software products, have enabled the researcher to make many different measures of uterine contractility over a period of time. These measurements include frequency of contractions, amplitude, maximum force of contractions, average force of contractions, integrals of contractile area under the curve, and the proportions of any measure of contractile activity observed in relation to a known agonist. In practice, most measurements focus on either amplitude or some measure of force (mean contractile force in a period of time, or integral of force).
This myriad of possible measurements leads to confusion in the review process, and there are hitherto minimal data comparing the efficacy or reliability of one measurement vs another. This pertains particularly to compounds that exert a uterotonic effect because the evaluation of the effects of an inhibitory compound can at least be related, by any of the numerous measurements, to the degree of contractile performance observed prior to exposure. However, for drugs that exert a uterotonic effect, evaluation of their effects on contractile activity is more challenging, with no clearly delineated methodology. Different authors use different techniques for assessment of such uterine contractile performance, and none of the techniques have been subjected to scrutiny in terms of objectivity, variability, and reliability.
The aim of this study was to evaluate the effects of 3 different uterotonic agents, oxytocin, the prostanoid TP receptor agonist U46619, and the α-adrenoceptor agonist phenylephrine, in tissue samples obtained from different patient donors, and within the same patient donor samples, and to investigate some commonly used measurement techniques. First, the use of the negative log of the molar concentration compound which exerts the half maximal effect (pEC 50 ) as a measurement of the sensitivity of both mean contractile force and amplitude of contraction was evaluated. Second, the reliability of the potassium chloride challenge, and its use in the expression of the maximal contractile activity achieved, for both mean contractile force and amplitude were studied. Finally, the variability of results, across tissue samples from different patient donors and from strips obtained from the same patient donors was investigated.
Materials and Methods
Tissue collection
Biopsy specimens of human myometrial tissue were obtained at elective cesarean section operations carried out at the Department of Obstetrics and Gynecology, University College Hospital Galway, Galway, Ireland. Biopsies were obtained from the myometrium in the midline, in the upper lip of the incision in the lower uterine segment as previously described. Ethics Committee (institutional review board) approval for tissue collection was obtained from the Research Ethics Committee at University College Hospital Galway, and recruitment of patients was by written informed consent.
The biopsies were placed in physiologic salt solution (PSS) on collection. The PSS contained the following ingredients: 4.7 mmol/L potassium chloride, 118 mmol/L sodium chloride, 1.2 mmol/L magnesium sulphate, 1.2 mmol/L calcium chloride, 1.2 mmol/L potassium phosphate, 25 mmol/L sodium bicarbonate, and 11 mmol/L glucose (Sigma-Aldrich, Dublin, Ireland). Immediately on collection, tissue biopsy specimens were placed in PSS at 4°C. Specimens were transported to the laboratory, transferred to PSS at room temperature, and used for experimentation within 8 hours of initial collection.
Tissue bath experiments
The biopsy specimens were dissected free of decidua and serosa, and 8 longitudinal (along the plane of the muscle fibres) myometrial strips were prepared measuring approximately 2 × 2 × 10 mm. The strips were mounted for isometric recording under 20 mN of tension in tissue baths as previously described. The baths contained 20 mL of PSS, pH 7.4, at a bath temperature of 37°C and gassed continuously with a mixture of 95% oxygen/5% carbon dioxide. When all strips had attained a steady 20 mN baseline, fresh PSS was introduced. Myometrial strips were allowed to equilibrate under these conditions for a period of at least 1 hour, after which they were challenged with potassium chloride (KCl) at a bath concentration of 30 mM. The KCl was left in contact with the tissue for a period of 10 minutes, followed by a PSS washout of the bath for a period of 10 minutes. This was then followed by 2 similar KCl challenges.
Following the washout with PSS after the third KCl challenge, the myometrial strips were exposed to cumulatively increasing concentrations of either oxytocin, the thromboxane A 2 mimetic U46619, or the α-adrenoceptor agonist phenylephrine. The initial bath concentration used for each compound was calculated after consideration of the known sensitivities of myometrial smooth muscle to that particular compound from earlier experiments and previous reports.
For all 3 compounds, there were 10 time points, each 12 minutes apart, at which cumulatively increasing doses were added to the tissue bath, each subsequent dose producing an approximate half-log unit increase in bath concentration compared with the previous dose administered. For oxytocin, the initial bath concentration achieved was 1 pmol/L, increasing to a final bath concentration of 44.4 nmol/L. For U44619, the initial bath concentration used was 28.5 pmol/L, increasing to a maximum bath concentration of 1.27 μmol/L. For phenylephrine, the initial bath concentration used was 10 nmol/L, increasing to a final bath concentration of 444 μmol/L.
Contractile activity measurements
All contractile activity was simultaneously recorded using the PowerLab hardware unit and the Chart version 4.0 software (AD Instruments, Hastings, UK). The maximum amplitude of contraction (MAMP) in, and the mean contractile force (MCF) for, the latter 10 minutes of the 12 minute exposure to a particular bath concentration of agonist were used as indices of the contractile activity in response to that particular concentration. Data were expressed as a percentage of the third KCl challenge by normalizing to the appropriate MAMP or MCF value.
The measurements were made in a total of 72 myometrial strips, which included 24 following exposure to each of oxytocin, U46619, and phenylephrine, all strips having been dissected from 9 patient donor samples. This allowed for data from all 9 patient donors in relation to all 3 agonists and provided results for 2-3 samples within a single donor (3 agonists among 8 baths) for each agonist. The measurements from the increasing bath concentrations enabled the construction of concentration-effect curves using the Solver Function in Microsoft Excel 2003 (SP3; Microsoft, Redmond, WA) to calculate various parameters of the response as described below.
Curve fitting and analysis
In cases in which the response to the agonist was monophasic, concentration-effect curves were constructed from the data obtained and fitted to a 1 receptor model using the following equation:
where E is the effect of the agonist, C is the molar concentration of the agonist, and pEC 50 is the negative log of the molar concentration of the agonist that produces a half-maximal response.
In cases in which the response appeared biphasic, an attempt was made to fit concentration-effect curves to a 2 receptor model using the following equation:
E = [ E max / ( 1 + ( 10 ^ ( − p EC 501 − log C 1 ) ) ) ] + [ E min / ( 1 + ( 10 ^ ( − p EC 502 − log C 2 ) ) ) ]
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