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The Contractile Effects of Oxytocin, Ergonovine, and Carboprost and Their Combinations

An In Vitro Study on Human Myometrial Strips

Balki, Mrinalini MBBS, MD*†; Erik-Soussi, Magda MSc*; Ramachandran, Nivetha PhD*; Kingdom, John MD*; Carvalho, Jose C. A. MD, PhD*†

doi: 10.1213/ANE.0000000000000682
Obstetric Anesthesiology: Research Report
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BACKGROUND: The objective of this study was to compare the in vitro contractile effects of the combination of oxytocin (low dose and high dose) with either ergonovine or carboprost in myometrial strips from women undergoing cesarean delivery (CD), and to study the effect of oxytocin pretreatment on these contractions. We hypothesized that the use of ergonovine or carboprost in combination with oxytocin would improve contractility compared with oxytocin alone.

METHODS: Myometrial samples obtained from women undergoing elective CD were pretreated in organ bath chambers with either oxytocin 10−5 M (experimental) or physiological salt solution (control) for 2 hours. They were then washed and subjected to dose-response testing with oxytocin, ergonovine, or carboprost (10−10 to 10−5 M), either alone or in combination with a fixed low-dose (10−10 M) (LDOx) or high-dose (10−6 M) (HDOx) oxytocin. The amplitude, frequency, area under the curve, and motility index (amplitude × frequency) of contractions during the dose-response period were analyzed with linear regression models, and compared among the groups. The primary outcome was the motility index across the study groups.

RESULTS: One hundred sixty-nine experiments were done in samples obtained from 56 women. The mean square root of the motility index [standard error] (√g·contractions/10 min) of oxytocin was significantly higher in the control (3.40 [0.24]) versus experimental group (2.02 [0.15]) (P < 0.001). When all control groups were compared, the motility index of oxytocin (3.21 [0.25]) was higher than that of ergonovine (2.13 [0.30], P < 0.001 [multiple comparisons adjusted P value, P < 0.001]), carboprost (1.88 [0.10], P < 0.001 [P < 0.001]), ergonovine + LDOx (2.07 [0.15], P < 0.001 [P < 0.001]), and carboprost + LDOx (1.82 [0.15], P < 0.001 [P < 0.001]) and was not different than that of ergonovine + HDOx (3.39 [0.32], P = 0.68 [P = 0.99]) and carboprost + HDOx (2.68 [0.30], P = 0.20 [P = 0.60]). However, in oxytocin-pretreated groups, carboprost + LDOx (motility index: 2.53 [0.08], P = 0.001 [multiple comparisons adjusted P value, P = 0.002]) and ergonovine + HDOx (2.82 [0.15], P < 0.001 [P < 0.001]) exhibited significantly superior contractility response compared with oxytocin alone, while ergonovine + LDOx (2.47 [0.13], P = 0.01 [P = 0.08]) and carboprost + HDOx (2.51 [0.20], P = 0.05 [P = 0.24]) showed higher mean contractility response compared with oxytocin alone but failed to reach statistical significance in adjusted analyses.

CONCLUSIONS: The attenuation of oxytocin-induced contractility in oxytocin-pretreated myometrial strips is in keeping with the previously established oxytocin-receptor desensitization phenomenon. Oxytocin is the most effective of the uterotonics tested if the myometrium is not preexposed to oxytocin. However, in the oxytocin-pretreated myometrium, a synergistic response is evident, and the combination of oxytocin with either ergonovine or carboprost produces superior response compared with oxytocin alone. Further in vivo studies in humans are necessary to determine whether these differences identified in vitro are clinically significant.

From the Departments of *Anesthesia and Pain Management and Obstetrics and Gynaecology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada.

Accepted for publication November 8, 2014.

Funding: This work was funded by Inaugural SOAP/Gertie Marx Research & Education Grant; and Department of Obstetrics and Gynaecology, Mount Sinai Hospital, University of Toronto. Dr. Mrinalini Balki was supported by funding from Canadian Anesthesiologists’ Society Career Scientist Award from the Canadian Anesthesia Research Foundation (CARF); University of Toronto Merit Awards, Department of Anesthesia; and Mount Sinai Hospital Foundation.

The authors declare no conflicts of interest.

This paper won the Zuspan Award at the 46th Annual Society for Obstetric Anesthesia and Perinatology Meeting, Toronto, Ontario, May 14–18, 2014. This study was also presented, in part, in the Richard Knill competition at the Canadian Anesthesiologists’ Society Annual Meeting, St. John’s, Newfoundland, June 13–16, 2014.

Address correspondence and reprint requests to Mrinalini Balki, MD, Department of Anesthesia and Pain Management, Mount Sinai Hospital, University of Toronto, 600 University Ave., Room 19-104, Toronto, Ontario M5G 1X5, Canada. Address e-mail to mrinalini.balki@uhn.ca.

Postpartum hemorrhage (PPH) is a leading cause of maternal morbidity and mortality, contributing to about 35% of maternal deaths worldwide.1 The incidence of PPH is increasing gradually despite advances in medical and surgical management.1,2 Since uterine atony is the most common cause, uterotonic therapy is the mainstay of PPH prevention and treatment. Oxytocin is commonly used as the first-line uterotonic agent, while other drugs such as ergonovine or carboprost are most commonly used in the event of persistently poor uterine tone when incrementally larger bolus and infusion doses of oxytocin have proven ineffective.3

Worldwide, there is a wide variability in published practice guidelines for the use of uterotonic drugs to prevent and treat PPH.4–7 The existing literature, including the Cochrane Collaboration of systematic reviews, is inconclusive when comparing the efficacies and potencies of various uterotonic drugs for the treatment of primary PPH.3,8–10 This, in part, could be related to the presence of several confounding factors influencing uterine tone and blood loss in the clinical setting of PPH, in addition to the heterogeneity in the trials and insufficient information on the outcome measures. Dose-response studies of the individual uterotonic drugs are scarce, and the interaction among them is still poorly understood.

Furthermore, the oxytocin receptor desensitization, which occurs with exogenous oxytocin exposure in labor, may lead to decreased responsiveness to oxytocin administration after delivery, resulting in excessive bleeding.11–15 This phenomenon is unlikely to affect the actions of other drugs acting through different pathways, such as ergonovine or carboprost.16,17 However, the effects of these drugs in combination with oxytocin in oxytocin preexposed myometrium have not been previously investigated.

The objective of this study was to compare the in vitro contractility of the combination of oxytocin (low dose and high dose) with either ergonovine or carboprost in myometrial strips from women undergoing cesarean delivery (CD), and to investigate the effect of oxytocin pretreatment on these contractions. We hypothesized that the use of ergonovine or carboprost in combination with oxytocin would improve contractility compared with oxytocin alone in myometrial strips, with or without oxytocin pretreatment.

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METHODS

The study was conducted as a prospective laboratory investigation after approval by the Research Ethics Board at Mount Sinai Hospital, Toronto, ON, Canada (REB 06-0095-E). The patient recruitment was done between May 2011 and July 2013. Written informed consent was obtained from all patients before their enrollment in the study. The inclusion criteria were nonlaboring women scheduled for elective primary or first repeat CD under spinal anesthesia at a gestational age of 37 to 41 weeks. We excluded women with previous uterine surgery other than a single previous CD, those requiring general anesthesia, women in labor undergoing emergency CD, previous history or any risk factors for PPH including abnormal placentation, multiple gestation, preeclampsia, macrosomia, polyhydramnios, uterine fibroids, bleeding diathesis, chorioamnionitis, or diabetes mellitus requiring insulin, and those taking medications that could affect myometrial contractility, such as labetalol, nifedipine, or magnesium sulfate. The study was registered at ClinicalTrials.gov (Registration number: NCT00989027; Principal investigator: Mrinalini Balki; Date: July 29, 2009).

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Reagents and Equipment

All drug solutions and their dilutions were prepared in sterile double-distilled water. Oxytocin (lyophilized powder) and ergonovine maleate were obtained from Sigma-Aldrich Canada Ltd., Oakville, ON, Canada. Oxytocin powder and reconstituted dilutions of the drug were stored at 4°C, and diluted ergonovine maleate drug solutions were stored in aliquots at –20°C and thawed on ice just before use. Carboprost, reconstituted in neat oil [15(S)-15-methyl prostaglandin F2α, the metabolically stable analog of PGF2α], was purchased from Cayman Chemical Company, Ann Arbor, MI, and stored at –20°C. Once reconstituted in sterile distilled water, the shelf life of carboprost is guaranteed for only 24 hours, and therefore fresh dilutions were prepared within several hours before each use. Further, vials containing carboprost dilutions were wrapped in aluminum foil to prevent exposure to light. All chemical constituents used in the preparation of 3-N-morpholino propane sulfonic acid (MOPS) buffer (145 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl2, 1.17 mM MgSO4·7H2O, 1.2 mM NaH2PO4·H2O, 3.0 mM MOPS solution, 5.0 mM glucose, and 2.0 mM pyruvate), physiological salt solution (PSS) (112 mM NaCl, 25 mM NaHCO3, 1 mM KH2PO4, 5 mM KCl, 1.2 mM MgSO4·7H2O, 11.5 mM glucose, and 2.5 mM CaCl2), and high potassium solution (120 mM KCl, 24 mM NaHCO3, 5 mM dextrose, 100 mM MgCl2, 1.2 mM KH2PO4, and 2 mM CaCl2) were obtained from Sigma-Aldrich, Oakville, ON, Canada.

A Radnoti 4-unit solution organ bath system (model no. 159920, purchased from Harvard Apparatus, Saint-Laurent, QC, Canada) was used for performing the contractility experiments. Carbogen (Praxair, Mississauga, ON, Canada) was used to continuously oxygenate the PSS in the organ baths throughout the experiment.

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Sample Biopsies and Preparation of Myometrial Strips

All patients in the study were administered spinal anesthesia. The obstetrician excised a sliver of myometrium of approximately 4 cm × 2 cm × 2 cm from the upper border of the uterine incision after the delivery of the fetus and placenta, but before the administration of intravenous oxytocin. The sample was collected in ice-cold MOPS buffer with a pH of 7.4 and transported immediately to the laboratory for performing contractility experiments. The buffer solution with sample was placed on ice for dissection and isolation of myometrial strips. Four longitudinal myometrial strips (2 mm × 2 mm × 10 mm each) were sectioned, mounted, and immersed in 10 mL of PSS in water-jacketed, temperature-controlled organ bath chambers, while attached to isometric force transducers by using clipped wire strings. The PSS in the organ bath system was maintained at pH 7.4, temperature 37°C, and continuously aerated with 95% oxygen and 5% carbon dioxide. The high potassium solution was preheated to 37°C in a water bath before commencing the experiment.

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Contractility Analysis

The mounted myometrial strips were allowed to equilibrate in PSS at 1 g tension for about 2 hours until spontaneous contractions were recorded as described previously.18 Myometrial strips that did not develop immediate spontaneous contractions were flushed with PSS every 10 to 15 minutes until stable regular spontaneous contractions were recorded for at least 25 minutes. After this equilibration period, each myometrial strip was exposed to high potassium solution, to examine the maximal contractile capacity of the tissue. The high potassium solution was drained from the baths, followed by 3 PSS washes to remove any residual solution.

Individual myometrial strips were then exposed to either oxytocin 10–5 M (experimental group) for a 2-hour period or left in PSS without oxytocin (control group).18 After this pretreatment period, the strips in both control and experimental groups were washed with PSS, equilibrated for 10 minutes in fresh PSS, and subjected to a dose-response testing with the study drugs. The dose-response testing was done for the following drugs, individually or in combination: (1) oxytocin, (2) ergonovine, (3) carboprost, (4) ergonovine with fixed low-dose (10−10 M) oxytocin (LDOx), (5) carboprost with fixed LDOx, (6) ergonovine with fixed high-dose (10–6 M) oxytocin (HDOx), and (7) carboprost with fixed HDOx (Table 1). The study drugs were administered cumulatively as 1 log molar concentration increase every 10 minutes from 10−10 to 10–5 M concentration, schematically presented in Figure 1. Because of the limited number of organ bath chambers, only 4 group assignments could be done at a time. Patients were assigned to either control or experimental groups alternatively, and all 4 strips from an individual patient were subjected to dose-response testing with 4 different study drug groups in a set sequence. All the failed experiments were noted, and make-up experiments were done in the end. A log book was maintained to track all experiments, and all electronic tracings were labeled with the group name.

Table 1

Table 1

Figure 1

Figure 1

The myometrial contractility was recorded via force transducers connected to a computer system running the AcqKnowledge 3.9.0 software with MP100 (Biopac System Inc., Goleta, CA). The baseline contractions were measured during the last 1500 seconds of the equilibration period, and those during each step of the dose-response period were measured for 600 seconds. At the end of the dose-response period, the strips were washed with PSS and once again exposed to the high potassium solution to assess muscle viability. The myometrial strips were removed from the apparatus, dried, and weighed. If samples failed to develop stable, measurable spontaneous contractions, or if there were any technical problems or experimental errors, the data sets were excluded from analysis.

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Statistical Analysis

The amplitude (g) and frequency (number of contractions in 10 minutes) of contractions were measured in each group, and the motility index (amplitude × number of contractions/10 min [g·number of contractions/10 min]) and the area under the force × time curve (AUC) (grams × seconds [g·s]) were derived to reflect the uterine activity and the strength of contractions, respectively.18 We compared various groups for the contractility parameters measured during the dose-response period.

The sample size was based on the availability of the patients. Usually, for basic science studies, a sample size of 6 per group is considered adequate. On the basis of our previous studies,14,16–18 a sample size of 9 to 12 per drug group was considered sufficient for this study.

We analyzed data with classic linear regression models (maximal likelihood method for parameters estimations) adjusted for repeated measures per sample (i.e., measurements at multiple concentrations) using a compound symmetry covariance structure.

The choice of which type of covariance structure to use in this study was data-driven. A number of potential different covariance structures were tested, and the model QIC (Quasi-Akaike Information Criterion) was used to choose the covariance structure with the best fit to data. On the basis of this criterion, a compound symmetry covariance structure was chosen. Once we determined that the compound symmetry covariance structure was the best fit, we compared the model QIC to that of a similar regression model where all observations were treated as being independent of each other (exchangeable correlation matrix) and confirmed that the adjustment for repeated measures through a compound symmetry covariance structure was appropriate and necessary in this case.

A compound symmetry covariance structure assumes that all observations on a given subject are equally correlated with each other. Generalized estimating equations were generated, including type of drug and patient group (control and experimental) during the dose-response period. Generalized estimating equation models allow for an unequal number of observations on each subject and reweighs observations through a covariance structure to obtain a balanced regression model in which each subject has the same statistical weight, regardless of the number of observations. This approach includes all study groups in a single regression model with single Wald χ2 determining the overall statistical significance of the exposure (modeled as a single categorical variable) and allowing the regression equation to compare any 2 exposures. Dunnett-Hsu correction was used to provide estimates of statistical significance when controlling for family-wise error rate in cases where multiple pairwise comparisons were derived from a single regression model; however, interpretation of data was based on actual effect-size estimates and their associated confidence interval.

The regression models used in this study have several assumptions: (1) that the outcome variable is linear, (2) that continuous covariates are also linear and have a consistent range with meaningful intervals, and (3) that the available sample size was sufficient to fit the regression model including all covariates and the covariance structure (all models with an observation per parameter ratio >10). Linearity of outcomes and continuous covariate was assessed through visual inspection of the data as was covariate range consistency. After comparing multiple transformation (visual inspection and model correlation coefficient), the outcomes were square-root transformed to adjust for the skewed distribution of both motility index and AUC. The model determined the effect of increasing concentration stratified by drug and was adjusted for covariates such as patients’ age, body mass index, gestational age, baseline tone during equilibration, baseline values for contraction parameters (amplitude, frequency, AUC, motility index) during equilibration, maximal amplitude after potassium chloride given at the beginning and end of dose-response curves, baseline tone before starting dose-response, and dry weight of sample. A sensitivity analysis was added to the main regression models in which no adjustments were performed other than baseline contraction values (without which the data are uninterpretable). The values were expressed as predicted mean and standard error or estimated differences (95% confidence intervals [CIs]). Visual inspection of residuals from regression models did not reveal any important deviation from a normal distribution or any association with study groups.

All statistical analyses were performed using SAS statistical software v9.2 (The SAS Institute, Cary, NC), and the statistician was blinded to various patient groups. A 2-tailed P value of <0.05 was considered statistically significant.

A total of 169 experiments were performed in samples obtained from 56 women (Fig. 2). The characteristics of patients included in the study are shown in Table 2.

Table 2

Table 2

Figure 2

Figure 2

The dose-response curves of motility index and AUC for individual drugs, including oxytocin, ergonovine, and carboprost in both control and experimental groups, are shown in Figures 3 and 4, respectively. When directly comparing control and experimental groups to each other, the motility index (regression model adjusted √mean [SE]) of oxytocin (all concentrations) was significantly attenuated in the experimental (2.02 [0.15] √g·contractions/10 min) as compared with the control group (3.40 [0.24] √g·contractions/10 min) (P < 0.001). However, there was no significant difference in the control versus experimental groups of ergonovine (2.32 [0.29] vs 2.20 [0.21] √g·contractions/10 min; P = 0.76) or carboprost (1.89 [0.09] vs 1.83 [0.22] √g·contractions/10 min; P = 0.79).

Figure 3

Figure 3

Figure 4

Figure 4

When all control groups were compared with each other in a single regression model, the motility index of oxytocin was significantly higher than that of ergonovine and carboprost (Table 3). Oxytocin also produced contractions with a significantly higher motility index than ergonovine + LDOx and carboprost + LDOx. However, there was no significant difference in the motility index of oxytocin versus either ergonovine + HDOx or carboprost + HDOx, nor was there any difference between ergonovine + HDOx and carboprost + HDOx (estimate: +0.71 [95% CI, –0.04 to +1.46] √g·contractions/10 min, P = 0.06 [adjusted P = 0.19]) (Table 3, Fig. 5).

Table 3

Table 3

Figure 5

Figure 5

When the experimental groups were compared, the motility index of ergonovine was higher than that of oxytocin; however, there was no significant difference in the response between oxytocin and carboprost (Table 4). The combination of ergonovine + LDOx, carboprost + LDOx, ergonovine + HDOx, and carboprost + HDOx exhibited a significantly superior contractility response when compared with oxytocin alone (Table 4, Fig. 5).

Table 4

Table 4

Figures 6 and 7 outline the dose-response curves of motility index and AUC, respectively, for high-dose and low-dose oxytocin combination groups derived from regression models adjusted as detailed in Statistical Analysis.

Figure 6

Figure 6

Figure 7

Figure 7

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DISCUSSION

Our in vitro study demonstrates that oxytocin pretreatment results in attenuation of myometrial contractility with subsequent administration of oxytocin, but not with ergonovine or carboprost. Furthermore, our results demonstrate that the combination of oxytocin with either ergonovine or carboprost is likely to be more effective under such circumstances. However, in oxytocin-naïve myometrium, oxytocin is still the most effective drug when compared with ergonovine or carboprost or their combinations. Our study findings are novel because dose-response profiles of various uterotonics or their combinations have not been investigated in either clinical or laboratory settings.

The observed attenuated response of oxytocin-induced contractility in myometrial strips pretreated with oxytocin confirms the previously established oxytocin-receptor desensitization phenomenon. In a previous study published by our group, oxytocin-induced contractility was found to be attenuated in a concentration- and time-dependent manner when the myometrial strips were pretreated with oxytocin 10−10, 10–8, or 10–5 M for 2, 4, 6, or 12 hours.18 The effect was predominantly seen with pretreatment with oxytocin 10–5 M for at least 2 hours or 10–8 M for at least 4 hours. This established desensitization model was used as a basis for studying various uterotonic drugs in the current study.

Our results are also in keeping with 2 of our previous studies.16,17 The first was conducted on isolated myometrial strips of pregnant rats.16 We demonstrated that the contractility induced by ergonovine or prostaglandin F2α remains unaffected by prior oxytocin exposure, indicating that the mechanism involved in desensitization does not interfere with these 2 uterotonics acting via different signaling pathways. The second was conducted on myometrial strips obtained from nonlaboring women or those with augmented labor. Ergonovine, prostaglandin F2α, and misoprostol produced similar contractions in both subsets, whereas oxytocin resulted in poor contractility in women with augmented labor.17

The molecular mechanism of oxytocin-induced desensitization is well understood; however, more research is needed in this area to understand the implications of this phenomenon. As a G-protein–coupled receptor, the oxytocin receptor undergoes rapid desensitization characterized by decreased cellular responsiveness and impaired signal transduction with continuous or repeated homologous stimulation. The phenomenon likely involves receptor phosphorylation, sequestration, internalization, and either degradation by lysosomes or recycling into the cell membrane.12,19 This is characterized by downregulation of oxytocin receptors and messenger RNA, as well as decrease in oxytocin binding.12,13 The clinical implications of the desensitization phenomenon can be significant, leading to failed induction of labor, failure to progress in labor requiring CD, and poor uterine responsiveness to oxytocin after delivery leading to uterine atony and PPH. With the current practice of administering increasingly larger doses of oxytocin over prolonged periods during induction or augmentation of labor, this phenomenon is highly relevant.

High-dose aggressive oxytocin protocols or prolonged administration of oxytocin for labor induction or augmentation are associated with a higher incidence of PPH related to uterine atony compared with low-dose regimens.15,20 In a previous study, we found that women with oxytocin-augmented labor require 9-fold greater doses of oxytocin (90% effective dose = 2.99 IU; 95% CI, 2.32–3.67 IU) to produce adequate uterine contractions during CD compared with the dose effective in nonlaboring women (90% effective dose = 0.35 IU; 95% CI, 0.18–0.52 IU).21,22 These clinical findings likely indicate differences in oxytocin receptor distribution or function, signaling pathways, and sensitivity to oxytocin in laboring and nonlaboring women, and clearly warrant the need for different management protocols in these settings.

There is limited clinical evidence in the literature on the comparative effects of individual uterotonic drugs or their combinations in women with or without augmented labor to suggest clear guidance to their use. In a 2013 Cochrane review of 5 trials (n = 2226) by Westhoff et al.,8 prophylactic oxytocin was found to be superior to ergot alkaloids in preventing PPH greater than 500 mL (relative risk 0.76; 95% CI, 0.61–0.94 mL); however, in subgroup analysis in which only randomized trials with low risk of methodologic bias were analyzed, this benefit did not persist. The authors suggested a lack of high-quality evidence supporting a benefit of prophylactic oxytocin over ergot alkaloids. The use of oxytocin was, however, associated with fewer side effects such as nausea and vomiting, making oxytocin the more desirable option for routine use to prevent PPH. The authors also found no benefit in the combination of oxytocin and ergometrine versus ergometrine alone (5 trials; n = 2891) in preventing PPH greater than 500 mL (relative risk 0.90; 95% CI, 0.34–2.41).8 Another review of 6 trials (n = 9332) by McDonald et al. found a small reduction in the risk of PPH (blood loss between 500 and 1000 mL) with a combination of oxytocin 5 IU and ergometrine 0.5 mg when compared with oxytocin 5 or 10 IU (odds ratio 0.82; 95% CI, 0.71–0.95). This difference was greater with the lower dose of oxytocin, but was not demonstrated for estimated blood loss of >1000 mL. Vomiting, nausea, and hypertension were more in the oxytocin–ergometrine combination.9 Tunçalp et al.,10 in their review of 13 trials comparing injectable prostaglandins with oxytocin or ergot alkaloids, found that the reporting of primary outcomes such as a blood loss of 1000 mL or more and the use of additional uterotonics was insufficient to give any reliable estimates. Because of the insufficient information in the existing clinical trials, our study findings have the potential to help design studies to better tailor prophylactic uterotonic regimens to specific clinical situations.

In our study, we found that among the 3 uterotonics, oxytocin is the most effective drug if the myometrium is not preexposed to oxytocin. Even when various combination groups (i.e., high-dose or low-dose oxytocin with ergonovine or carboprost) were compared with oxytocin alone, the effect seemed to be mainly driven by the presence of oxytocin during the dose-response period. The response with HDOx-ergonovine or HDOx-carboprost combination was similar to that of oxytocin alone, which implies that at a certain high concentration of oxytocin, most receptors are saturated with oxytocin and a maximal possible contractile response is produced exclusively with oxytocin. Under such circumstances, there is no benefit of adding ergonovine or carboprost to oxytocin to further enhance contractility. By contrast, poor contractility seen with the addition of low-dose oxytocin to ergonovine or carboprost compared with oxytocin alone suggests that the amount of oxytocin in these combination groups was not sufficient for the available oxytocin receptors to induce effective myometrial contractility. Based on our results, it appears to be desirable to increase the dose of oxytocin until maximal response is produced, especially in nonlaboring women undergoing CDs. However, one should be cautious, because higher doses may be associated with side effects.

In the oxytocin-pretreated myometrium, among the 3 individual uterotonics, ergonovine produces the most effective contractions followed by oxytocin and then carboprost. This relative difference is not due to improved contractility of ergonovine, but rather to the reduced efficacy of oxytocin in the desensitized myometrium. However, it is unlikely that ergonovine will be used as a primary sole agent for prophylaxis of PPH in women exposed to oxytocin during labor, considering its greater potential for side effects compared with oxytocin. Nevertheless, it is evident that in the myometrium previously exposed to oxytocin, contractility induced by oxytocin alone is not adequate; it is lower than all combination groups, and the addition of ergonovine or carboprost is required to produce a synergistic response. This can be explained by the desensitization phenomenon, resulting in attenuation of oxytocin-induced contractility response, thereby rendering the response produced by other uterotonic drugs superior. It is likely that with oxytocin pretreatment, the downregulation of receptors limits the binding of oxytocin to functioning myometrial oxytocin receptors and results in attenuated response, regardless of the administered dose. This implies that ergonovine or carboprost should be considered either prophylactically or in the event of poor uterine responsiveness to oxytocin, especially if the uterus is preexposed to oxytocin during labor.

There are some limitations to our study, including the in vitro study design, which may not exactly replicate clinical findings. Unlike our previous report, we did not observe a typical increase in contractility response with the increasing doses of uterotonics. The reason for this finding is unclear and may be related to the variable number of myocytes within a sample or to the variability in the genetic make-up of different individuals. The concentrations used in the study were chosen based on our previous research and standard concentrations used in most dose-response studies in the literature.11,14,16–18 There is insufficient information in the literature regarding the plasma levels of uterotonics after parenteral administration in the setting of PPH. The reported levels of oxytocin vary significantly during pregnancy, labor, and postpartum from 10–12 to 10–8 M and may not accurately reflect local myometrial concentration.23–26 The serum levels of ergonovine after oral administration were found to be in the range of 10–6 to 10–18 M; however, these were measured after variable clinical doses in a population in which gender and pregnancy status were not clearly described.27–29 The approximate peak serum levels of carboprost after IM administration of 250 μg range from 2718 to 3097 pg/mL (10–8 M) in term pregnant women undergoing vaginal deliveries.30 Our cumulative dose response in the range of 10−10 to 10−5 M likely reflects the levels reached after parenteral administration of these drugs postpartum. However, it should be noted that in vitro concentrations may not reflect the in vivo plasma levels and perhaps represent an overestimation in the experimental settings. It is unclear whether the addition of higher concentrations of ergonovine and carboprost to oxytocin than those used in our study will have any additive effect. However, clinically, the use of ergonovine and carboprost is likely to be associated with more side effects.

In summary, we have confirmed that oxytocin pretreatment attenuates contractility induced by oxytocin, but not by ergonovine and carboprost. In oxytocin-naïve myometrium, oxytocin is the most effective drug and there is no benefit to adding ergonovine or carboprost once the maximal dose of oxytocin is given. In oxytocin-pretreated myometrium, however, a combination of oxytocin with ergonovine or carboprost produces a superior response than oxytocin alone. On the basis of our findings, we suggest the choice of uterotonic drug for prevention of PPH should be based on labor type. However, further in vivo studies are warranted to confirm these findings and to determine whether these differences observed in vitro are clinically significant.

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DISCLOSURES

Name: Mrinalini Balki, MBBS, MD.

Contribution: This author is responsible for the design and conduct of the study, data collection and analysis, and writing and revision of the manuscript.

Attestation: Mrinalini Balki attests to the integrity of the original data and the analysis reported in this manuscript, and is the archival author responsible for maintaining the study records.

Name: Magda Erik-Soussi, MSc.

Contribution: This author helped in the design and conduct of the study, data collection and analysis, and manuscript revision.

Attestation: Magda Erik-Soussi attests to having reviewed the original data and the analysis reported in this manuscript and approved the final manuscript.

Name: Nivetha Ramachandran, PhD.

Contribution: This author helped in the conduct of the study, data collection and analysis, and manuscript preparation.

Attestation: Nivetha Ramachandran attests to the integrity of the original data and the analysis reported in this manuscript, and approved the final manuscript.

Name: John Kingdom, MD.

Contribution: This author helped in the study design and conduct, data analysis, and manuscript revision.

Attestation: John Kingdom attests to having reviewed the original data and the analysis reported in this manuscript, and approved the final manuscript.

Name: Jose C. A. Carvalho, MD, PhD.

Contribution: This author helped in the design and conduct of the study, data collection and analysis, and manuscript preparation.

Attestation: Jose C. A. Carvalho attests to having reviewed the original data and the analysis reported in this manuscript, and approved the final manuscript.

This manuscript was handled by: Cynthia A. Wong, MD.

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ACKNOWLEDGMENTS

The authors acknowledge Cedric Manlhiot, PhD(c), Clinical Research Program Manager, Labatt Family Heart Centre, The Hospital for Sick Children, Toronto, for statistical analysis of this study. The authors also thank Ms. Kristi Downey (Research Coordinator) for assisting with patient recruitment, and Dr. Stephen Lye (Associate Director, Research) and Dr. Lee Adamson (Senior Investigator) from the Samuel Lunenfeld Research Institute, Toronto, for their continued guidance and support for our research.

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