Postpartum hemorrhage (PPH) remains a major cause of maternal morbidity and significant mortality.1 The incidence of this obstetrical emergency is increasing worldwide2 and is largely attributed to an increase in uterine atony.1–4
A significant risk factor for uterine atony is oxytocin exposure during labor, resulting in decreased effectiveness of subsequent oxytocin administration. Exposure to large doses of oxytocin for prolonged periods during labor is associated with severe PPH secondary to uterine atony.5 Women who received exogenous oxytocin during labor require 9-fold greater doses of oxytocin6 during cesarean deliveries to produce adequate postpartum uterine tone when compared with their oxytocin-naive nonlaboring counterparts.7
On a molecular level, this phenomenon is referred to as oxytocin receptor (OTR) desensitization. The OTR is a G-protein–coupled receptor (GPCR). All GPCRs undergo receptor desensitization after agonist stimulation, a protective physiological response to prevent overstimulation of the receptor, resulting in inactivation. In most cases, with elimination of the agonist, the GPCR responsiveness is recovered by the process of resensitization.8
Despite significant advances in unravelling the mechanisms, mediators, and pathways of GPCR desensitization, little is known about GPCR resensitization. There is sufficient evidence to suggest that the process of resensitization is critical to the regulation of GPCRs and their cell signaling.9 Although recovery of the OTR after desensitization is complex, we postulate that resensitization is a time-dependent process after removal of its agonist, oxytocin. Thus, withdrawal of oxytocin for a specific interval after oxytocin-induced desensitization may allow for adequate resensitization and recovery of the OTR, improving the responsiveness of subsequently administered oxytocin.
The objective of this study was to investigate the recovery time course of oxytocin sensitivity in human myometrium in vitro after oxytocin-induced desensitization. We hypothesized that there would be a positive correlation between an increased duration of recovery period and subsequent oxytocin sensitivity, leading to superior myometrial contractility after longer, compared with shorter, recovery intervals.
This prospective laboratory investigation was undertaken after approval by the Research Ethics Board at Mount Sinai Hospital, Toronto, Ontario, Canada (MSH REB # 13-0261-A, dated November 28, 2013). Written informed consent was obtained from participating nonlaboring term pregnant women undergoing elective cesarean delivery between December 2013 and June 2014. The study was registered with the ClinicalTrials.gov registry (NCT02051231) before patient enrollment.
The inclusion criteria were nonlaboring women with gestational age 38 to 41 weeks requiring a primary or first repeat cesarean delivery under spinal anesthesia. We excluded women with previous uterine surgery (other than a single previous cesarean delivery), requirement for general anesthesia, those taking any medication that could affect uterine contractility, or any condition predisposing to uterine atony or PPH, such as multiple gestation, abnormal placentation, diabetes mellitus requiring insulin, preeclampsia, polyhydramnios, macrosomia, uterine fibroids, chorioamnionitis, coagulopathy, or a history of PPH.
All drug solutions were prepared by serial dilutions in sterile double-distilled water. All salts and reagents used in the preparation of 3-N-morpholino propanesulfonic acid solution (MOPS) and physiological salt solution (PSS), as well as oxytocin (lyophilized powder) were obtained from Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada. The PSS (112 mM NaCl, 25 mM NaHCO3, 1 mM KH2PO4, 5 mM potassium chloride [KCl], 1.2 mM MgSO4.7H2O, 11.5 mM glucose, and 2.5 mM CaCl2) and MOPS buffer solution with a pH of 7.4 (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, 5.0 mM glucose, and 2.0 mM pyruvate) were prepared in advance and stored at 4 to 8°C.10,11
Myometrial Strip Isolation and Preparation
The patients underwent a lower segment cesarean delivery after initiation of spinal anesthesia. After delivery of the neonate, and before oxytocin administration, a small sliver of myometrium from the upper border of the incision on the lower uterine segment was excised by the obstetrician. The sample of myometrium was immersed in ice-cold MOPS buffer solution. Within 15 minutes, 4 myometrial strips, each of 10 mm × 2 mm × 2 mm dimensions, were dissected along the longitudinal direction of the muscle fibers, and each strip suspended vertically in an individual temperature-controlled organ bath chamber containing 10 mL PSS at pH 7.4. Homeostatic conditions were maintained in the organ bath solution, including temperature control at 37°C and continuous aeration with a mixture of 95% oxygen and 5% carbon dioxide. Each strip was clipped at the upper end to an isometric force transducer (Radnoti 4 Unit Tissue Organ Bath System, model 159920; Harvard Apparatus Canada, Saint Laurent, Quebec, Canada) and at the lower end to the base of the organ bath chamber.
Individual myometrial strips were allowed to equilibrate in PSS at 1 g tension and maintain regular spontaneous contractions for up to 2 hours, as per our previous investigations (Fig. 1).11 Myometrial strips not developing immediate spontaneous contractions were flushed with PSS every 10 to 15 minutes until regular contractions were obtained for at least 30 minutes. After equilibration, to establish the maximal contractile capacity of the strips, each strip was exposed to 120 mM KCl. The KCl was then drained, and to ensure full removal of residual KCl, the bath was washed with PSS 3 times.
Individual strips were pretreated with oxytocin 10−5 M concentration (experimental groups) for 2 hours to induce OTR desensitization. After pretreatment, the strips were washed with PSS 3 times. The myometrial strips were allowed to rest in the PSS solution for 30, 60, or 90 minutes. At the end of the rest period, all strips were subjected to dose-response testing with oxytocin 10−10 to 10−5 M, increased cumulatively in a pattern of 1 log molar concentration every 10 minutes. At the end of the dose-response test, the strips were washed with PSS and subjected to a final KCl 120 mM solution to assess tissue viability. The strips were removed from the baths, manually dried, and weighed. In addition to the 3 experimental groups, 1 control group was studied in parallel, whereby there was no oxytocin pretreatment, and no rest period before the oxytocin dose-response.
To control for any unidentified variation in the organ bath conditions, the study groups were sequentially rotated across the various organ baths. A logbook was maintained to track all the experiments, and all electronic tracings were labeled with the study group name.
Continuous measurement and recording of amplitude and frequency of myometrial contractions was performed by force displacement transducers connected to a data acquisition system with AcqKnowledge® 3.9.0 software, MP 100 (Biopac System Inc., Goleta, CA). If a strip did not develop any spontaneous contractions, or produced contractions of approximately <0.2 g above baseline during the equilibrium period, it was considered to have poor or failed contractions. The experiments with failed/poor myometrial contractions during equilibration, no contractions during dose-response, or with technical errors were excluded from the analyses.
The amplitude (g) and frequency (number of contractions per 10 minutes) of contractions were recorded during the equilibration period and during each step of dose-response curve. The motility index (amplitude × frequency; g·contractions/10 min) and area under the force × time curve (AUC; g·s) of contractions were measured and analyzed to reflect uterine activity and the strength of contractions, respectively.10–12 The primary outcome was the motility index, whereas the secondary outcomes included amplitude, frequency, and AUC. After comparing multiple potential mathematical transformations (visual inspection and correlation coefficient of the model), the outcomes were transformed by square root to adjust for the skewed distribution of both motility index and AUC.
Amplitude, frequency, AUC, and motility index were analyzed with linear regression models (maximal likelihood method for parameters estimation) adjusted for repeated measures (i.e., measurements performed at multiple concentrations) per sample using a compound symmetry covariance structure. The primary assumption of the compound symmetry covariance structure is that all observations on a given subject are equally correlated with each other. In previous studies using similar methodology,10–12 a data-driven approach established the need to adjust for repeated measures and determined that compound symmetry was the optimal choice.
Several assumptions were relevant to the regression models used in this study. First, the linearity of the outcome variables and of continuous covariates with meaningful intervals and a defined range was confirmed through visual inspection of the outcomes and continuous covariates. The second assumption was that the available sample size was sufficient to fit all necessary covariates and the covariance structure in the regression model using a threshold of observation per parameter ratio >10.
Generalized estimating equations (GEE) were generated during the dose-response period. GEE 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. The main regression models for this study included all study groups in the same ordinal variable as the study exposure. The statistical significance of the difference between the study groups was determined using Wald χ2. Dunnett-Hsu test was used to recalculate confidence intervals (CIs) and P values after adjusting for family-wise error rate. Results were interpreted based on Dunnett-Hsu corrected values.
Models for each contractile parameter were adjusted for baseline tone, amplitude, frequency and AUC during equilibration, weight of sample, maximal amplitude after KCl administered before pretreatment and at the end of dose-response, as well as the basal tone before the first step of the dose-response. The values were expressed as study group–specific predicted means and standard error (SE) or estimated differences (95% CI) of the summation of measurements captured over the range of oxytocin concentrations from 10−10 to 10−5 M during the dose-response period of 60-minute duration. Finally, residuals from the regression models were not associated with study group and had no important deviation from a normal distribution. A 2-tailed P value of <0.05 was considered statistically significant. All statistical analyses were performed using SAS statistical software v9.2 (The SAS Institute, Cary, NC).
The sample size for this study was based on historical sample size for similar studies.10–12 Usually for basic science studies, a sample size of 6 per group is considered adequate. On the basis of our previous studies, a sample size of 10 to 12 experiments per drug group was considered sufficient for this study.
Patient recruitment for this study was done from December 2013 to June 2014. A total of 41 women were approached for participation in the study and 22 consented (Fig. 2). Myometrial samples were obtained from 20 women, yielding a total of 80 experiments. Of these, 55 experiments, done on samples from 16 patients, were successful and used for analysis (Fig. 2). The characteristics of patients included in the study are shown in Table 1. The mean (SE) values of the contractile parameters, estimated from the adjusted regression models, in various study groups during the dose-response period are presented in Table 2.
The estimated differences (95% CI) between groups from type 3 GEE regression model are shown in Table 3 for the adjusted values as well as for values corrected for family-wise error rate using the Dunnett-Hsu test. The motility index of contractions (√g·contractions/10 min) was significantly lower in 30-, 60-, and 90-minute groups compared with the control group. The AUC levels (√g·s) were also significantly lower in 30- and 90-minute groups compared with control values. Figure 3 demonstrates the dose-response curves of all contractile parameters with increasing concentrations of oxytocin for each study group.
Contractile parameters among the experimental groups did not reveal a significant difference for any of the pairwise comparisons; however, the CIs for the differences in mean values were wide. The mean estimated difference of motility index of contractions (√g·contractions/10 min) after Dunnet-Hsu adjustment for 30 vs 60 minute was 0.27 (95% CI, −0.82 to 1.35; P = 0.58), 30 vs 90 minute was 0.56 (95% CI, −0.53 to 1.64; P = 0.27), and 60 vs 90 minute was 0.29 (95% CI, −0.80 to 1.38; P = 0.55).
This in vitro study shows that oxytocin pretreatment of human myometrial strips attenuates oxytocin-induced contractility, despite a rest period of up to 90 minutes after oxytocin administration. However, we were unable to determine whether increasing the rest period from 30 to 90 minutes results in improvement in myometrial contractility because of our small sample size relative to the variability in the contractile parameters. Thus, the experimental design did not allow us to confirm our hypothesis, as we predicted a longer rest period would allow recovery of the desensitized OTR and subsequent superior myometrial contractility.
The OTR is a GPCR and therefore susceptible to desensitization.8 Stimulation by its agonist, oxytocin, results in desensitization of the receptor. In clinical terms, this can lead to decreased uterine effectiveness of subsequent oxytocin administration, resulting in failure of labor progression, as well as uterine atony in the immediate postpartum phase. Oxytocin exposure during labor is an independent risk factor for severe atonic PPH.5
The classic model of desensitization focuses on phosphorylation of the GPCR as a principal mechanism. This is undertaken by kinase enzymes, also known as GPCR kinases (GRKs). After GRK phosphorylation, regulatory proteins, known as β-arrestins, bind to the phosphorylated complex to uncouple it from the G-protein, deactivating the G-protein. Internalization of the phosphorylated GPCR complex occurs as it is targeted to clathrin-coated pits, and it is guided to either lysosomes for degradation or endosomes where the GPCR is resensitized via dephosphorylation and recycled to the cell membrane to function once again (Fig. 4).8
Several studies have demonstrated that the length of time of oxytocin pretreatment to myometrial cells or myometrial tissue can determine the extent of OTR desensitization.11,13–16 In human myocytes, 4.2 hours of oxytocin pretreatment leads to a 50% reduction in OTR activity, with complete inactivity after 6 hours of pretreatment.13 Myometrial tissue in vitro has reliably demonstrated that oxytocin pretreatment of 10−5 M of 2 hours or 10−8 M of 4 hours leads to significantly attenuated contractility.11 Given that the duration of oxytocin pretreatment can affect the extent of OTR desensitization, it is certainly feasible that the time of rest after removal of oxytocin pretreatment may affect the extent of OTR resensitization, perhaps facilitating recovery and recycling of the OTR. This could lead to an increased responsiveness of the uterine smooth muscle to subsequent oxytocin and therefore remains a critical topic to explore in the prevention of PPH.
Phaneuf et al.14–16 have repeatedly demonstrated that the binding of oxytocin is reduced and the OTRs remain nonfunctional after desensitization; however, the number of OTRs is, in fact, unchanged. This suggests that pretreatment does not cause degradation of the receptor and that there is potential for OTR resensitization to increase receptor availability and functioning at the cell surface.
There is also evidence to indicate that a longer rest period after pretreatment appears to enhance the function of the OTR in cell-based studies. A longer rest period of 4 hours (versus 45 minutes) results in a marked increase in oxytocin-OTR binding,17 and a 15-minute rest period (versus 5-minutes) leads to an increase in effective oxytocin responsiveness.18
The mechanism of OTR resensitization after a rest period is more likely to be recycling of receptors as opposed to degradation and synthesis of the new OTR. The number of OTRs available for binding at the cell surface after pretreatment is similar in the presence of cycloheximide, a protein synthesis inhibitor, indicating that de novo receptor synthesis is not the major mechanism of new OTRs at the cell surface.17 This is further supported by the fact that, after internalization of the OTR, intracellular trafficking in human embryonic kidney cells is not directed to degradation organelles (i.e., lysosomes).17 It should be noted that the study was done in cultured myometrial cells from hysterectomy specimens and not from the uterine samples from pregnant women.
Our observations did not demonstrate any difference in the recovery of myometrial tissue after a rest period of 30 to 90 minutes, but, unfortunately, the study was underpowered. Given that our study investigated contractile outcomes of myometrium, it is not possible to reliably comment on the molecular aspects of the OTR to account for these results. It is possible that in this in vitro experiment, the OTRs are directed to degradation pathways as opposed to recycling pathways, a hypothesis that has been previously inferred.19 If this is the case, it is currently not clear how long the process of OTR resynthesis would take. However, the literature to date regarding OTR resensitization is mostly in favor of recovery of the receptors with fully functioning recycled OTRs at the cell surface.17 The current literature is restricted to cell-based studies,14–17 with differences in the patient population, study designs, and assessment methods among studies. It has been suggested that perhaps the OTR regulation differs in its native tissue and in in vitro conditions.18 Furthermore, studies of myometrial cells from the pregnant population13–15 show differences in desensitization of OTRs compared with nonpregnant tissue.17–19 The expression of the enzyme GRK6 (an isoenzyme of GRKs) has been reported to increase at term pregnancy. Overexpression of GRK6 has been associated with an increase in OTR desensitization and a delay of recovery, whereas GRK6 knockdown attenuates OTR desensitization.18 These factors could explain the observed lack of improved responsiveness in the contractile properties of term pregnant desensitized myometrium with a specified rest period when compared with nonpregnant cell-based studies, which demonstrate full functioning recovery of OTRs after pretreatment and a longer rest period.
It is also possible that although resensitization changes occur at the molecular and receptor level, they are simply not translated into observable changes in myometrial contractility, which is obviously important when investigating atonic PPH. Perhaps, some factor other than time is responsible for resetting the OTR. The present study is the first to look at the time of recovery of the OTR in human pregnant myometrium through measurements in the strength and frequency of contractions, which are key determinants of uterine tone.
There are several limitations of our investigation. First, in retrospect, the study was underpowered; we chose the sample size based on previous experiments. Second, an in vitro human myometrial model for oxytocin-induced desensitization may not be suitable for exploring resensitization. Although our in vitro model reliably produces OTR desensitization, it may not provide the conditions that support OTR resensitization. Possible reasons may be that recycling of receptors or generation of new receptors may be a process that depends on gene expression and biosynthesis of mediators and proteins,20 and an in vitro model in an organ bath may not necessarily provide the optimum medium that in vivo conditions could.
We hypothesized that the longer duration of recovery time would promote maximal resensitization. Theoretically, the myometrial strips resting for 90 minutes are at higher risk of compromise in their tissue viability than those resting for only 30 minutes, and therefore, any benefit from a longer rest period may have been negated. Because of a limitation of the number of organ baths, we were unable to run control on nonpretreated groups with rest periods of 30 to 90 minutes, which could have addressed this potential tissue viability bias.
Perhaps, a resting time longer than 90 minutes is required, and our longest rest time of 90 minutes may not have been sufficient to permit adequate OTR recovery. Conti et al.17 showed a marked increase in OTR binding at the cell surface with a 4-hour rest period compared with a 45-minute rest period. The rest periods of 30, 60, and 90 minutes in our experimental groups were selected based on time frames that would be feasible in a clinical environment for proceeding to cesarean delivery in an oxytocin-augmented parturient with arrest of labor. Investigating longer periods of rest, such as 4 hours, is unlikely to be both feasible and relevant in a clinical context, and therefore, because of limited organ bath availability, this was not investigated. Conversely, it is possible that the maximal beneficial recovery time is shorter than 30 minutes and, in hindsight, a pretreated group allocated to a shorter rest time would have helped address this.
In summary, the results of the present study do not allow us to determine whether a rest period of up to 90 minutes after oxytocin-induced desensitization in human myometrial strips in vitro leads to improved oxytocin-induced contractility. It is imperative to conduct further investigations, including further experiments to elucidate key mechanisms to the pathway of OTR recovery. Clinical in vivo studies assessing the association between the time interval from cessation of oxytocin to cesarean delivery in the augmented labor arrest population, and the assessment of uterine tone and estimated blood loss in the immediate postpartum phase may provide further information on the relationship between uterine responsiveness and previous exposure to oxytocin.
Name: Mrinalini Balki, 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: Nivetha Ramachandran, PhD.
Contribution: This author helped in the design and conduct of the study, data collection, data analysis, and manuscript preparation.
Attestation: Nivetha Ramachandran attests to having reviewed the original data and the analysis reported in this manuscript and approved the final manuscript.
Name: Sang Lee, MD.
Contribution: This author helped in the design and conduct of the study, data collection, data analysis, and manuscript preparation.
Attestation: Sang Lee attests to having reviewed the original data and the analysis reported in this manuscript and approved the final manuscript.
Name: Chiraag Talati, MBBS, BSc (Hons), FRCA.
Contribution: This author helped in the conduct of the study, data collection, data analysis, and manuscript preparation.
Attestation: Chiraag Talati 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.
The authors acknowledge Cedric Manlhiot, PhD, 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 design of figures; Ms. Alice Luca (Research Technician) for guidance; 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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