A role for adrenergic receptors in the uterotonic effects of ergometrine in isolated human term non- laboring myometrium Rebecca A Fanning, MD, FCAI, MSc, Florike Sheehan, BSc, oxytocin in myometrium previously exposed to oxytocin15, 16. Despite Claire Leyden, BSc, Niamh Duffy, MSc, Luis F. Iglesias-Martinez, the long standing use of ergometrine, the exact mechanism by which it MSc, Michael F Carey, MD, Deirdre P Campion, PhD, John J. exerts its uterotonic effect in human tissue has never been fully O’Connor, PhD. elucidated. Its administration has been linked with an increase in 1Department of Perioperative Medicine, Coombe Women and Infants University Hospital, frequency of myometrial contractions corresponding with increased Cork Street, Dublin 8, Ireland 2UCD School of Biomolecular and Biomedical Science, UCD Conway Institute of basal tone. The increase in basal tone is thought to be due to an effect Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland on the inner layer of the myometrium, as this layer is rich in 3Systems Biology Ireland, University College Dublin, Dublin 4, Ireland 4 adrenoceptors, and reacts to epinephrine administration with an UCD School of Veterinary Medicine, University College Dublin, Dublin 4, Ireland increase in tonus and frequency of contractions17,18. The mechanism of action of ergometrine has been linked to stimulation of human α1 ABSTRACT adrenoreceptors19, 20. In this study we have investigated the effects of a Background: Ergometrine is a uterotonic agent that is recommended number of adrenoceptor ligands on the action of ergometrine on in the prevention and management of post partum hemorrhage. isolated human myometrial smooth muscle. Despite its long-standing use the mechanism by which it acts in humans has never been fully elucidated. The objective of this study METHODS was to investigate the role of adrenoreceptors in ergometrine’s Subjects mechanism of action in human myometrium. The study examined the The study was conducted as a prospective laboratory investigation. hypothesis that alpha adrenoreceptor antagonism would result in the Biopsy specimens of human pregnant myometrial tissue were reversal of the uterotonic effects of ergometrine. obtained from non-laboring women undergoing elective lower Methods: Myometrial samples were obtained from women segment cesarean delivery. All patients were ASA physical status 2 undergoing elective cesarean delivery. The samples were then pregnant women and gave written informed consent. Ethical approval dissected into strips and mounted in organ bath chambers. Following -15 for the study was obtained from the Research Ethics Committee of the generation of an ergometrine concentration-response curve (10 to Coombe Women and Infants University Hospital (2006-22). All 10-5M), strips were treated with increasing concentrations of -15 -7 -7 -5 samples were collected during the four months from September to ergometrine (10 to 10 M) alone and ergometrine (10 to 10 M) in December in each of 2011, 2012, and 2013. Women with a singleton the presence of phentolamine (10-7 M), prazosin (10-7 M), propranolol -6 -6 gestation at 38-40 weeks who were not in labor prior to their cesarean (10 M) or yohimbine (10 M). The effects of adding ergometrine and delivery were considered for inclusion. Exclusion criteria included the effect of drug combinations were analysed using linear mixed ultra-sound findings consistent with fetal IUGR, polyhydramnios or effects models with measures of amplitude (g), frequency oligohydramnios, history of a chronic medical condition in pregnancy (contractions/10min) and motility index (g*contractions/10min). or pregnancy related condition requiring the use of medication, history Results: A total of 157 experiments were completed on samples of ruptured membranes, a diagnosis of human immunodeficiency obtained from 33 women. There was a significant increase in the virus, hepatitis B or C, suspected abnormal placentation and a booking motility index (adding 0.342 g*counts/10min/µM; 95% CI from 0.253 BMI >30 kg/m2. Indications for cesarean delivery included breech to 0.431, P<0.001), amplitude (0.078 g/µM; 95% CI, from 0.0344 to presentation and prior cesarean delivery. All patients received antacid 0.121, P=5e-04) and frequency (0.051 counts/10min/µM; 95% CI, prophylaxis with 30 ml of 0.3 M sodium citrate and 400 mg 0.038 to 0.063, P<0.001) in the presence of ergometrine. The α cimetidine orally preoperatively and a spinal anesthetic with 2.0 to 2.4 adrenergic antagonist phentolamine and the more selective α1 ml 0.5% hyperbaric bupivacaine, with 20 to 25 microgram (mcg) of adrenergic antagonist prazosin, inhibited the ergometrine mediated intrathecal fentanyl and 100 to 150 mcg of intrathecal morphine. increase in motility index, amplitude and frequency (-1.63 Oxytocin (Novartis Pharmaceuticals UK Ltd., Horsham, West Sussex, g*counts/10mins/µM and -16.70 g*counts/10mins/µM for motility UK) 5IU was administered by slow intravenous bolus following index, respectively). delivery of the baby and cord clamping. Conclusions: These results provide novel evidence for a role for α adrenergic signaling mechanisms in the action of ergometrine on Tissue preparation human myometrial smooth muscle in the in vitro setting. Information The myometrial biopsy was excised from the midline of the upper that sheds light on the mechanism of action of ergometrine may have margin of the lower uterine segment incision (inner myometrial layer) implications for the development of further uterotonic agents. following delivery of the baby and placenta. All specimens were thoroughly rinsed in Ringer’s lactate solution ensuring all traces of Key Words: ergometrine, human myometrium, phentolamine, blood were removed and that the specimen was free of placental prazosin, propanolol, alpha adrenergic receptors. tissue. For tissue bath experiments the biopsies were placed in a sterile container and refrigerated at 4ºC until used, which was within 2 to 20 Corresponding Author Dr. John J. O’Connor, , UCD School of Biomolecular and Biomedical Science, Conway h of collection. Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland., +353 1716 6765, [email protected] Contractility Analysis Biopsies were dissected into longitudinal muscle strips of INTRODUCTION approximately 12 by 5 by 2 mm. Isometric tension recordings were Powerful and efficient myometrial contractions and retractions are obtained from an eight-chamber organ bath (10 ml, water jacketed) essential to compress the uterine vasculature arresting blood flow system (Myobath, World Precision Instruments Inc. Sarasota, from the placental bed after delivery of the foetus and placenta1. Florida). The organ baths contained Krebs-Henseleit physiological Failure of the uterus to contract leads to uterine atony and postpartum salt solution (PSS; NaCl 118 mmol/l, D-glucose 11.1 mmol/l, haemorrhage (PPH). PPH remains a common cause of both maternal NaHCO3 24.9 mmol/l, MgSO4 1.2 mmol/l, KCl 4.7 mmol/l, KH2PO4 2-4 morbidity and mortality . Evidence is emerging of increasing rates of 1.2 mmol/l, and CaCl2 2.5 mmol/l, pH 7.4) and were aerated with a 2, 5-8 PPH worldwide which has largely been attributed to an increased gas mixture of 95% O2 and 5% CO2 and maintained at 37 ºC. incidence of uterine atony2, 7, 9, 10. Using prophylactic uterotonic agents Myometrial strips were then allowed to equilibrate at 1 to 2 grams (g) compared to conservative management lowers maternal blood loss tension until a steady tension was achieved as previously described21. and reduces the risk of PPH11. Oxytocin is commonly used as the first During this equilibration period, the Krebs solution was changed line utertonic agent but if the uterus fails to contract adequately every 10 min. When spontaneous contractions became regular (within current practice guidelines recommend the use of additional utertonic 60-90 min) the amplitude (g) and frequency (contractions/10min) agents to prevent and treat PPH such as ergometrine, misporostol and were recorded as follows: (1) a 30 min control period followed by carboprost12-14. Availability of pharmacological agents that exert their cumulatively increasing concentrations of ergometrine every 30 min effects through alternative receptor mechanisms is extremely relevant from 10-15 M to 10-8 M (n=14) or 10-7 M to 10-5 M (n=22) and (2) a 30 in light of emergencing evidence of decreased responsiveness to min control period followed by the addition of antagonist and then 30 min later by cumulatively increasing concentrations of ergometrine contraction measurements by ergometrine in comparison to the from 10-7 M to 10-5 M. A time matched control strip from each patient, variance in the data that is unexplained by neither ergometrine nor exposed only to Krebs solution was run in parallel with each separate patient specific effects24,25. Given samples available from 23 patients, experiment to ensure tissue viability for the duration of the post hoc power calculation indicated that the available data was experiment. In addition a time-matched control for antagonists alone sufficient to identify an effect size of f-squared = 0.05 with 83% (over 120 min) was also carried out. The motility index (amplitude x power. This N was chosen to cover concentrations of ergometrine frequency; g*contractions/10min) was calculated to determine the from 10-15 M to 10-10 M and in a separate experiment, 15 samples from uterine activity and the strength of contractions with the use of the 10-9 M to 10-5 M. No individual experiment was duplicated in any one Powerlab software, Chart (version 5.0; AD Instruments Pty Ltd, Bella patient tissue. The analysis was performed using the PWR package in Vista, NSW, Australia). The primary outcome was the motility index R. of myometrial contractions induced by ergometrine, epinephrine or antagonists. Secondary outcomes included amplitude and frequency Effect of ergometrine combined with the other drugs parameters. As in the previous section, for Figures 4 and 5 we compared two LMEs to determine the effect of drugs in combination with Reagents ergometrine on contraction measurements independently.