In Vitro Comparative Effect of Carbetocin and Oxytocin in Pregnant Human Myometrium with and without Oxytocin Pretreatment

OXYTOCIN remains the first-line uterotonic agent for the prevention and treatment of postpartum hemorrhage (PPH) secondary to uterine atony. Despite its routine use worldwide, oxytocin has limitations that include hemodynamic side effects and need for infusion dosing due to its short half-life.^1–3  Furthermore, it has reduced effect in women who have undergone labor augmentation.^4,5  This has led to a search for newer agents for the prevention of PPH with greater efficacy and fewer side effects.

Carbetocin (1-deamino-1-carba-2-tyrosine(O-methyl)-oxytocin), a long-acting synthetic analog of oxytocin, was developed as an alternative to oxytocin and has been used in clinical practice for the last 2 decades. The Society of Obstetricians and Gynecologists of Canada recently updated their practice guideline to specifically recommend carbetocin over oxytocin infusion for the prevention of PPH during elective cesarean deliveries (CDs).⁶ 

One of the potential advantages of carbetocin over oxytocin is its longer half-life (4 to 10 times that of oxytocin), allowing for single bolus dosing rather than the continuous infusion or multiple doses of oxytocin in the postpartum period.^7,8  Despite limited data, it appears to have a similar side effect profile to that of oxytocin.⁹  The data on the comparative effects of carbetocin versus oxytocin for the prevention and treatment of PPH are inconsistent in the existing literature.^6,10–14  The limited evidence based on in vitro myometrial contractility studies in rats and humans suggests that carbetocin is less potent than oxytocin, thus requiring dose adjustments for clinical efficacy.^15–17 

Oxytocin preexposure has been shown to cause desensitization of oxytocin receptors in vitro in cultured human myocytes¹⁸  and to attenuate oxytocin-induced myometrial contractions in pregnant rats¹⁹  and humans.^20,21  This phenomenon is likely responsible for the high incidence of uterine atony and PPH in women with oxytocin-augmented labors.^5,22,23  None of the existing clinical or in vitro studies have yet examined the effect of oxytocin preexposure on contractile efficacy of carbetocin.

The purpose of this study was to compare the effects of oxytocin and carbetocin in human term pregnant myometrium in vitro, with and without oxytocin pretreatment. We hypothesized that oxytocin would induce stronger contractions than carbetocin and that oxytocin pretreatment would attenuate the myometrial contractility induced by both drugs.

After approval by the Research Ethics Board, Mount Sinai Hospital, Toronto, Ontario, Canada (Research Ethics Board No.: 11-0257-E, dated December 6, 2011), this prospective laboratory study was conducted at Mount Sinai Hospital and the Samuel Lunenfeld Research Institute in Toronto, Ontario, Canada, from December 30, 2011 to September 3, 2013 (ClinicalTrials.gov registry NCT01689298; principal investigator: M.B., Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada). The study included nonlaboring singleton pregnant women at 37 to 41 weeks gestational age undergoing elective primary or first repeat CD under spinal anesthesia. Written informed consent was obtained from all women enrolled in the study. Exclusion criteria were laboring women, those with previous multiple CDs or other myometrial surgeries, CD under general anesthesia, placental abnormalities, and other risk factors for PPH.

The attending anesthesiologist administered spinal anesthesia using 0.75% hyperbaric bupivacaine 1.8 ml with fentanyl 10 μg and epimorph 100 μg, as per routine in our hospital with standard monitoring. The obstetrician performed the CD via Pfannenstiel incision. After delivery of the fetus and placenta, a small sliver of myometrium (approximately 2 cm × 1 cm × 0.5 cm; weight 0.02 g) was excised from the upper margin of the lower segment transverse uterine incision, just before the administration of oxytocin. The uterus was then closed in a routine manner. The collected specimen was immediately placed in 3-N-morpholino propanesulfonic acid buffer solution (145 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl2, 1.17 mM MgSO4.7H2O, 1.2 mM NaH2PO4.H2O, 3.0 mM 3-N-morpholino propanesulfonic acid solution, 5.0 mM glucose, and 2.0 mM pyruvate) with pH 7.4. The endometrium and serosa were discarded if seen during dissection. The myometrial sample was then divided longitudinally into four strips of equal area, 10 mm × 2 mm × 2 mm each, parallel to the direction of the muscle fibers.

As previously described,^20,21  for the contractility analysis, the myometrial strips were mounted individually in four organ bath chambers (Radnoti four unit tissue-organ bath system, model 159920; Harvard Apparatus Canada, Canada) filled with 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) at 37°C and pH 7.4, to mimic physiological conditions. The organ bath solution was aerated continuously with a mixture of 95% O2 and 5% CO2 (carbogen). An initial resting tension of 1 g was applied to each strip. The myometrial strips were allowed to equilibrate until 25 min of continuous, spontaneous rhythmic contractions developed (usually within 2 h of mounting). If the spontaneous contractions did not start immediately, the PSS was changed by flushing every 10 to 15 min until spontaneous contractions were observed. After equilibration, the myometrial strips were stimulated with 120 mM KCl to provide both viability verification of the strips and a reference maximal contraction for analysis. The KCl solution was drained from the organ baths, and any residual solution was removed by washing three times with PSS.

Two of the four myometrial strips were pretreated with oxytocin 10⁻⁵ M (experimental group) and the other two were bathed in PSS (control group) for 2 h as a proxy for the laboring and nonlaboring uterus, respectively. This method is based on the tested model of our earlier study, which showed a significant attenuation of oxytocin-induced myometrial contractions after pretreatment with oxytocin 10⁻⁵ M for 2 h.²⁰  After the pretreatment period, the tissues were flushed three times to wash off all oxytocin in the bath. Thereafter, two strips, one from the experimental (oxytocin pretreated) and one from the control group, were subjected to dose–response testing with increasing concentrations of carbetocin in a pattern of 1 log molar increase every 10 min, from 10⁻¹⁰ to 10⁻⁵ M. The remaining two strips were similarly tested with increasing concentrations of oxytocin from 10⁻¹⁰ to 10⁻⁵ M (fig. 1). Myometrial contractions were continuously recorded using an isometric force transducer connected to a data acquisition system with AcqKnowledge^® 3.9.0 software, MP 100 (Biopac System Inc., USA). At the end of the experiment, a final stimulation with 120 mM KCl confirmed the viability of the myometrial strips (fig. 1).

The amplitude (g) and frequency (number of contractions in 10 min) of contractions were recorded for 1,500 s during the equilibration period and for 600 s during each step of the dose–response period. The motility index (amplitude × frequency; g.contractions/10 min) and the area under the curve (integral force; g.seconds) were calculated to determine the uterine activity and the strength of contractions, respectively.²⁰  The contractile parameters were compared among the groups.

The primary outcome was the motility index of myometrial contractions induced by oxytocin or carbetocin. Secondary outcomes included all other contractile parameters.

The data were analyzed with linear regression models (maximum likelihood method for parameter estimations) and adjusted for repeated measures per sample (i.e., measurements at multiple concentrations) through a compound symmetry covariance structure. 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 the 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 reweights 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. The outcome parameters were square-root transformed to adjust for their skewed distribution.^20,21  The models determine the effect of increasing concentration stratified by drug and were adjusted using various covariates such as patient characteristics (age, body mass index, and gestational age), baseline tone and contraction parameters (amplitude, frequency, area under the curve, and motility index) during equilibration, maximum amplitude after KCl given at the beginning and end of the dose–response testing, and dry weight of sample. The values were expressed as predicted mean and standard error. The half maximal effective concentration (EC50) was estimated based on pharmacodynamic modeling (four-parameter dose–response curve) and compared across various groups. Statistical analysis was carried out using SAS statistical software version 9.2 (The SAS Institute, USA), and the statistician was blinded to various study groups. A two-tailed P value of less than 0.05 was considered statistically significant.

Based on our previous studies, a sample size of 9 to 12 patients per drug group was considered sufficient for this study.^20,21 

Fifty-six women were approached for participation in the study and 20 of them consented, yielding 20 myometrial samples. Each myometrial sample was divided into four strips for a total of 80 strips. Of these 80 experiments, 17 were excluded for failure to contract and human/apparatus error, resulting in 63 successful experiments with similar distribution of strips across the four study groups. The study flow chart with patient recruitment and sample distribution is shown in figure 2.

The clinical characteristics of the participants are shown in table 1. The mean (SD) age of the patients was 36.1 (2.8) yr, body mass index was 28.7 (4.9) kg/m², and gestational age at delivery was 39.1 (range, 37.4 to 40.9) weeks. None of the participants experienced any complications including PPH up to 24 h postpartum.

The baseline values of contractile parameters during equilibration in all treatment groups, along with mean sample weights, are shown in table 2. Table 3 shows the contractile parameters induced by increasing concentrations of oxytocin and carbetocin in both control and experimental groups. After adjustment for all covariates, in the control groups, the predicted mean (standard error) motility index of contractions produced by oxytocin (3.01 [0.36] √g.contractions/10 min) across all concentrations from 10⁻¹⁰ to 10⁻⁵ M was found to be superior to that of carbetocin (2.16 [0.14] √g.contractions/10 min) (regression-estimated difference, 0.857 [95% CI, 0.290 to 1.425] √g.contractions/10 min; P= 0.003). Similarly, oxytocin-pretreated experimental strips produced stronger motility index of contractions with oxytocin (1.97 [0.20] √g.contractions/10 min) compared with carbetocin (1.16 [0.22] √g.contractions/10 min) during the dose–response period (0.813 [0.328 to 1.299] √g.contractions/10 min; P = 0.001).

Oxytocin pretreatment resulted in a significant reduction in motility index of contractions in the oxytocin group (−1.040 [−1.998 to −0.082] √g.contractions/10 min; P = 0.03) as well as in the carbetocin group (−0.996 [−1.392 to −0.560] √g.contractions/10 min; P < 0.001) (table 3) compared with their respective controls.

The estimated EC50, based on pharmacodynamic modeling, showed a similar trend in the findings although the differences between the groups were not significant. The EC50 (95% CI) for oxytocin was 10^−7.3 M (10^−7.7 to 10^−6.8 M) in control and 10^−6.5 M (10^−7.1 to 10^−5.9 M) in experimental group, whereas that for carbetocin was 10^−8.9 M (10^−13.0 to 10^−4.7 M) for control and 10^−7.0 M (10^−11.1 to 10^−3.0 M) for experimental group.

The dose–response curves of motility index for both oxytocin and carbetocin demonstrated a statistically significant gradient at concentrations 10⁻⁸, 10⁻⁹, and 10⁻¹⁰ M. The concentration in the motility index model has a chi-square value of 13.4 (degrees of freedom = 5) and P = 0.02. The square-root transformation makes curves appear flatter than they actually are.

The primary regression models for this study treated both study drug and study group as the two independent bilevel variables rather than a single variable with four distinct study groups. The pairwise inferences (described in the Results section) are derived from the contrasts of these two separate variables. Considering the parameterization approach, a P value adjustment was not necessary. However, recognizing that a different approach would have required adjustment, we reran the model using a four group design with Dunnett–Hsu adjustment and found the same results. The box plots in figure 3 show the motility index of carbetocin-induced and oxytocin-induced contractions in oxytocin-pretreated and control groups.

The results of our in vitro study show that in term pregnant human myometrium with no prior in vivo exposure to exogenous oxytocin, oxytocin produces stronger contractions than carbetocin over the entire studied range of equimolar concentrations and that oxytocin pretreatment attenuates the contractions produced by both drugs.

In vitro studies comparing carbetocin and oxytocin are scarce in the literature, with only three studies published so far: two in rat^15,16  and one in human myometrial samples.¹⁷  Atke and Vilhardt¹⁵  demonstrated in nonpregnant rat myometrium that the uterotonic activity of carbetocin was approximately 30 times smaller than that of oxytocin. Engstrøm et al.¹⁶  compared carbetocin and oxytocin over a dose–response range of 10⁻⁹ to 10⁻⁶ M in nonpregnant rat myometrium and found the maximum contractile effect of carbetocin was approximately 50% lower than that of oxytocin. However, these studies in nonpregnant myometrium do not account for pregnancy-associated changes in oxytocin signaling or receptor modulation. Norström et al.¹⁷  demonstrated the need for a fivefold higher concentration of carbetocin than oxytocin (10 × 10⁻¹⁰ M vs. 2 × 10⁻¹⁰ M, respectively) to produce defined contractions in human term pregnant myometrium; however, the study did not quantitatively measure the strength of these contractions. Of note, the sample size was small in all three studies (four to six cases each) and the concentration of treatment drugs varied across studies, making it difficult to quantify their effects on contractility. Moreover, the effect of oxytocin-induced desensitization on contractions was not examined.

Our findings are in agreement with these previous studies, showing that oxytocin produces stronger contractions than carbetocin at similar concentrations. Furthermore, our study design and larger sample size allowed us to compare the effects of carbetocin and oxytocin in pregnant human myometrium over a wide range of similar concentrations using multiple contractile parameters and evaluate the effect of oxytocin pretreatment on the uterotonic activity of both drugs.

Existing clinical evidence supports the efficacy of carbetocin for the prevention of PPH in CD, but research supporting this evidence is limited and requires further validation.^10,14  In 2009, the Society of Obstetricians and Gynecologists of Canada revised their guideline for the prevention and treatment of PPH in elective CD, recommending carbetocin 100 μg given as an IV bolus over 1 min rather than continuous oxytocin infusion.⁶  More recently in 2012, the Cochrane Database of Systematic Reviews¹⁰  studied the 11 existing clinical trials comparing carbetocin to conventional uterotonics (oxytocin or syntometrine) or placebo for prevention of PPH. Out of these trials, four studies compared carbetocin to oxytocin at CD, demonstrating that carbetocin was associated with a decreased need for additional uterotonics (relative risk [RR], 0.62; 95% CI, 0.44 to 0.88), reduced need for uterine massage (RR, 0.54; 95% CI, 0.37 to 0.79), and reduced risk of PPH (RR, 0.55; 95% CI, 0.31 to 0.95), but no significant difference in the average estimated blood loss.^7,11–13  As noted by the authors, this review was limited by several potential sources of bias, a small number of included studies, and heterogeneity of study populations across the trials. Furthermore, nonuniform oxytocin dosing was compared with a standard dose of carbetocin administered intravenously or intramuscularly in these studies.¹⁰ 

Since the publication of the 2012 Cochrane Review,¹⁰  three additional clinical trials have been published comparing carbetocin and oxytocin.^24–26  Two of these studies showed a decreased need for additional uterotonic drugs with carbetocin versus oxytocin at CD, similar to the Cochrane study.^24,25  The third study, however, found no difference between the two drugs in composite variable endpoint of blood loss above 1,500 ml, transfusion, hemoglobin reduction of 4 g/dl or more, or operative intervention.²⁶  As in prior trials, heterogeneity among these studies and their study populations as well as overall lack of randomization make the results difficult to interpret conclusively.

Although the literature comparing carbetocin to oxytocin is modest, an apparent contradiction is emerging between existing in vivo and in vitro evidence. Clinical studies suggest carbetocin may be either equally or more effective than oxytocin at elective CD,^{7,11,13,24–26}  whereas laboratory studies, including the current study, have shown that carbetocin appears to be less potent than oxytocin at similar concentrations. This paradox could be related to the use of nonequipotent doses of these drugs in clinical practice. Although the relative potencies of these drugs, when administered at delivery, have not been described so far, there is evidence that carbetocin 100 μg produces similar contractions to oxytocin 10 μg (equivalent to 5 U).²⁷ 

An alternative explanation for the discrepancy between in vitro and in vivo studies of carbetocin versus oxytocin might be related to the pharmacokinetics of carbetocin. Its increased lipophilicity,¹⁵  tissue distribution, and resistance to peptidase degradation contribute to a prolonged elimination half-life in vivo.⁸  The extended action of carbetocin may, in turn, decrease the need for additional uterotonics, thereby reducing the risk of uterine atony that accompanies the use of additional uterotonics.

Oxytocin receptor desensitization after oxytocin preexposure is an important phenomenon that has been well documented in the literature both in vivo and in vitro in human myometrium.^18,28,29  The mechanism of desensitization is not fully understood, but it is known to involve receptor down-regulation as well as a decrease in oxytocin receptor mRNA expression and oxytocin-binding sites.^18,30  The end result after prolonged exposure to oxytocin may be the diminished tissue responsiveness to additional oxytocin and poor myometrial contractility.^18,20  This was demonstrated in the contractility studies by Balki et al.,²⁰  which found attenuation of oxytocin-induced contractions after pretreatment with oxytocin 10⁻⁵ M for at least 2 h. This phenomenon has been supported by poor uterine contractility and increased blood loss at delivery after administration of high doses or prolonged exposure to oxytocin during labor.^4,21,22,31 

The effect of oxytocin preexposure on carbetocin-induced contractions was largely unknown until recently. Our in vitro study suggests that, similar to oxytocin, the attenuation of myometrial contractions after oxytocin preexposure is also seen with carbetocin. Because carbetocin is a partial oxytocin receptor agonist/antagonist^8,16  with similar binding affinity as oxytocin,¹⁶  oxytocin receptor desensitization may be the underlying mechanism for these observations with clinical implications that may be extrapolated. If carbetocin-induced contractility is attenuated by oxytocin preexposure in the clinical setting, similar concentrations of carbetocin may be less effective to prevent PPH in women with prolonged or oxytocin-augmented labor compared with women undergoing elective CD, and these women may require dose adjustments. However, the lack of monotonic response with carbetocin in laboring women and the high rate of arrhythmias with doses higher than 120 μg questions the utility of this drug in laboring women.³² 

There are some limitations to this study. Although we found significant differences in motility index between control and oxytocin-pretreated groups, such differences were not found to be significant when compared through EC50, despite a similar trend in the findings. Hence, we suggest that our observations should be interpreted with caution. Furthermore, experiments performed in vitro may not account for multiple clinical variables affecting myometrial contractility and blood loss. However, the absence of these external variables allows for a clear comparison of carbetocin and oxytocin in human myometrium while minimizing confounders. Direct translation of the concentrations used in this study to clinical doses may not be possible without further studies. We chose these concentrations based on standard concentrations used in most dose–response studies in the literature and our previous research findings.^18,20,21  Oxytocin plasma levels have been determined at delivery, but a wide range has been measured during labor, between 10⁻¹² and 10⁻⁸ M.^33–36  In elective CD, Yamaguchi et al.³⁷  observed plasma levels of 1.16 × 10⁻⁹ M, 4.07 × 10⁻¹⁰ M, and 6.15 × 10⁻⁹ M after oxytocin infusions of 10 U/30 min (0.33 U/min), 10 U/3 min:45 s (2.67 U/min), and 80 U/30 min (2.67 U/min), respectively. They suggested that plasma oxytocin concentrations may not accurately correlate with uterine activity, with local uterine concentrations possibly correlating more directly. Serum levels of carbetocin during labor and delivery have yet to be studied. Finally, similar to oxytocin, biomolecular studies may be required with carbetocin to understand the underlying mechanisms of our results and to confirm whether the attenuation of carbetocin-induced myometrial contractions after oxytocin preexposure is related to the oxytocin desensitization phenomenon.

In summary, we have compared the contractile effects of carbetocin and oxytocin in human pregnant myometrium in vitro and demonstrated that oxytocin is superior to carbetocin in producing contractions at similar concentrations. We have further demonstrated a reduction in contractility induced by oxytocin after oxytocin preexposure, with an even greater reduction in carbetocin-induced contractility after oxytocin preexposure, likely as a function of the oxytocin desensitization phenomenon. It is important to note that the half-life, protein binding, and effect on the receptor are significant clinical parameters that modulate each drug’s indications. Additional studies are warranted for clinical correlation with these findings.

The authors thank Stephen Lye, Ph.D., and Lee Adamson, Ph.D., Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada, for their guidance and collaborative support during the experiments. The authors also thank Cedric Manlhiot, Ph.D., Department of Pediatrics, Labatt Family Heart Centre and Hospital for Sick Children, Toronto, Ontario, Canada, for lending his statistical expertise to the data analysis.

This study was supported by Merit Awards from the Department of Anesthesia and Pain Management, University of Toronto, Toronto, Ontario, Canada.

The authors declare no competing interests.

Full protocol available from Dr. Balki: mrinalini.balki@uhn.ca. Raw data available from Dr. Balki: mrinalini.balki@uhn.ca.