Myometrial Strips Research Articles

Pheophorbide-a, a chlorophyll derivative, inhibits myometrial contractility ex vivo via blockade of extracellular and intracellular Ca2+−entry

BackgroundPreterm delivery is associated with neonatal complications such as neurological impairment and necrotising enterocolitis. Thus, suppression of early myometrial contractility in pre-term labor (PTL) delays delivery and reduces maternal adverse outcomes associated with PTL. However, most tocolytics exert varied efficacies and untoward outcomes such as maternal cardiovascular and respiratory effects creating a need for novel efficacious and safe tocolytics. We previously reported that chlorophyll derivatives including Pheophorbide-a (PBa) isolated from the leaves of the plant Ficus exasperata, effectively inhibited non-pregnant mouse myometrial contractility ex vivo. Therefore, the current study explores the mechanisms involved in PBa-induced myometrial contractility inhibition in both non-pregnant and pregnant mouse uterus. MethodWe carried out contractility studies by inducing isometric tension on isolated myometrial strips of mice following ex vivo exposure to PBa. The uterine strips were mounted in organ baths containing oxygenated physiological saline maintained at pH 7.4 and 36.8 ± 0.2 °C. ResultsPBa (0.02–206.19 µM) produced concentration-dependent decreases in the amplitude and frequency of pregnant and non-pregnant uterine contractions. In addition, PBa (87.74 μM) significantly inhibited the amplitude (P = 0.0026) and frequency (P = 0.0178) of OT-induced uterine contractility in Ca2+−free solution and in the presence of high KCl (80 mM) (P = 0.005). Furthermore, our study showed that the uterine relaxant effect of PBa occurred through the blockade of l-type voltage-gated Ca2+channels (VGCCs) and through inhibition of Ca2+ mobilization from intracellular stores which are common downstream pathways specific to tocolytics and relevant for uterine relaxation. ConclusionThese findings reveal that PBa effectively attenuates uterine contractions via interaction with Ca2+−entry and shows potential as a promising candidate as a tocolytic therapy for PTL. Further investigations on PBa are encouraged to elucidate molecular and cellular mechanisms of PBa uterine relaxation activity including assessment of efficacy and off-target effects.

Insights into the role of PGF2α in canine periparturient myometrium.

Parturition in dogs is subjected to complex hormonal regulation, with the involvement of prostaglandin F2α (PGF2α) still not fully understood. To investigate uterine inertia (UI), the most prevalent maternal reason for dystocia in the bitch, a better understanding of undisturbed uterine, especially myometrial function, is crucial. Our aim was to gain deeper insights into the role of PGF2α in the canine parturient myometrium. Uterine biopsies were obtained during medically indicated cesarean sections. To test for stimulatory effects of PGF2α in vitro, circular and longitudinal myometrial layer tissue strips were challenged with 50pM, 0.5µM, and 50µM PGF2α. Prostaglandin-endoperoxide synthase 2 (PTGS2) and PGF2α-receptor (PTGFR) mRNA expressions were compared between primary UI (PUI) and obstructive dystocia (OD) samples in isolated parturient myometrium. PTGFR protein expression was assessed in full thickness uterine samples. PGF2α concentrations were analyzed in canine interplacental tissue around term. In the organ bath, the contractile response to PGF2α was limited to the circular layer at the highest dosage. Correspondingly, PTGFR immunohistochemical staining was significantly stronger in the circular layer (p ≤ 0.01). PTGS2 gene expression did not differ between PUI and OD, whereas PTGFR gene expression could not be quantified. Local uterine PGF2α concentrations correlated negatively with serum P4 levels and were the highest during prepartum luteolysis while being significantly lower in PUI. Conclusively, despite the significant increase in local PGF2α concentrations at birth, confirming the interplacental tissue as a production site, our results suggest that PGF2α might affect uterine contractility during labor, mainly indirectly.

Extracellular acidification increases uterine contraction in pregnant mouse by increasing intracellular calcium.

As uterine extracellular pH decreases during the ischemic conditions of labor, but its effects on myometrial contraction are largely unknown, there is a need to elucidate its physiological effects and mechanisms of action. Furthermore, it is not known if any of the effects of extracellular acidification are affected by pregnancy, thus we also determined how gestation affects the response to acidification. Nonpregnant, mid-, and term-pregnant myometrial strips were obtained from humanely killed mice. Contractions were recorded under spontaneous, depolarized, and oxytocin-stimulated conditions. The extracellular pH of the perfusate was changed from 7.4 to 6.9 or 7.9 in HEPES-buffered physiological saline. Intracellular pH was measured using SNARF, and intracellular calcium was measured using Indo-1. Statistical differences were tested using the appropriate t-test. Extracellular acidification significantly increased the frequency and amplitude of spontaneous contractions in pregnant, but not nonpregnant, myometrium, whereas alkalinization decreased contractions. Intracellular acidification, via Na-butyrate, transiently increased force in pregnant tissue. Intracellular pH was gradually acidified when extracellular pH was acidified, but extracellular acidification increased contractility before any significant change in intracellular pH. If myometrial force was driven by oxytocin or high-K depolarization, then extracellular pH did not further increase force. Intracellular calcium changes mirrored those of force in the spontaneously contracting pregnant myometrium, and if calcium entry was prevented by nifedipine, extracellular acidification could not induce a rise in force. Extracellular acidification increases excitability, calcium entry, and thus force in pregnant mouse myometrium, and this may contribute to increasing contractions during labor when ischemic conditions and acidemia occur.

β3 Receptor Signaling in Pregnant Human Myometrium Suggests a Role for β3 Agonists as Tocolytics.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the β2 adrenergic receptor relax airway smooth muscle and are effective in the treatment of asthma led to the notion that β2 mimetics would prevent preterm birth by relaxing uterine smooth muscle. The activation of cAMP-dependent protein kinase by β2 receptors is unable to provide meaningful tocolysis. The failure of β2 agonists such as ritodrine and terbutaline to prevent preterm birth suggests that the regulation of uterine smooth muscle is disparate from that of airway. Other smooth muscle quiescent-mediating molecules, such as nitric oxide, relax vascular smooth muscle in a cGMP-protein kinase G-dependent manner; however, nitric oxide activation of protein kinase G fails to explain the relaxation of the myometrium to nitric oxide. Moreover, nitric oxide-mediated relaxation is blunted in preterm labor, and thus, for this reason and because of the fall in maternal blood pressure, nitric oxide cannot be employed as a tocolytic. The β3 adrenergic receptor-mediated relaxation of the human myometrium is claimed to be cAMP-dependent protein kinase-dependent. This is scientifically displeasing given the failure of β2 agonists as tocolytics and suggests a non-canonical signaling role for β3AR in myometrium. The addition of the β3 agonist mirabegron to pregnant human myometrial strips in the tissue bath relaxes oxytocin-induced contractions. Mirabegron stimulates nitric oxide production in myometrial microvascular endothelial cells, and the relaxation of uterine tissue in vitro is partially blocked by the addition of the endothelial nitric oxide synthase blocker Nω-Nitro-L-arginine. Recent data suggest that both endothelial and smooth muscle cells respond to β3 stimulation and contribute to relaxation through disparate signaling pathways. The repurposing of approved medications such as mirabegron (Mybetriq™) tested in human myometrium as uterine tocolytics can advance the prevention of preterm birth.

The In Vitro Contractile Response of Canine Pregnant Myometrium to Oxytocin and Denaverine Hydrochloride.

In pregnant bitches, the response to oxytocin and denaverine hydrochloride in dystocia management is usually poor. To better understand the effect of both drugs on myometrial contractility, the circular and longitudinal muscle layers were examined in an organ bath. For each layer, three myometrial strips were stimulated twice, each with one of three oxytocin concentrations. The effect of denaverine hydrochloride was studied once in direct combination with oxytocin and alone with subsequent oxytocin administration. Contractions were recorded and evaluated for average amplitude, mean force, area under the curve (AUC), and frequency. Effects of different treatments were analyzed and compared within and between layers. In the circular layer, oxytocin significantly increased amplitude and mean force compared to untreated controls regardless of stimulation cycles or concentrations. In both layers, high oxytocin concentrations caused tonic contractions, while the lowest concentration created regular rhythmic contractions. Longitudinal layer tissue responded to oxytocin with a significantly decreased contractility when stimulated twice, presumably a sign of desensitization. Denaverine hydrochloride neither affected oxytocin induced contractions nor showed a priming effect to subsequent oxytocin. Thus, no benefit of denaverine hydrochloride on myometrial contractility was found in the organ bath. Our results suggest a better efficiency of low-dose oxytocin in canine dystocia management.

β3 adrenergic receptor activation modulates connexin 43 activity to relax human myometrium

Preterm labor, delivery prior to 37 completed weeks of gestation, is the leading cause of infant morbidity and mortality. β3 adrenergic receptor protein expression is increased in the myometrium during pregnancy, and the agonist, mirabegron, relaxes the myometrium making the β3 adrenergic receptor a potential therapeutic target in PTL. β3 adrenergic receptor has been shown to activate the tyrosine kinase, Src, which can down regulate connexin 43, a contractile associated protein which promotes the formation of gap junctions that create an electrical syncytium. We hypothesize that mirabegron downregulates connexin 43, imparting quiescence effects on the myometrium. Employing contractile studies, we demonstrate that Src is involved in the mirabegron-induced relaxation of contracting pregnant human myometrial tissue strips. Western blot analysis demonstrates that Src kinase expression is decreased in both preterm and term laboring myometrial tissue. Imaging revealed that mirabegron stimulation of the β3 adrenergic receptor phosphorylates tyrosine at position Y265 on connexin 43 in pregnant human uterine myocytes. Western blot analysis and immunofluorescent imaging indicate that mirabegron decreases the expression of connexin 43 and mediates relaxation over a 24-h exposure period, suggesting that mirabegron has long lasting quiescent effects on the human myometrium. The relationship between the β3 adrenergic receptor and down regulation of the contractile associated protein connexin 43 through activation of Src kinase suggests that mirabegron may be useful in combination tocolysis.

Modulation of Melatonin Receptors Regulates Reproductive Physiology: The Impact of Agomelatine on the Estrus Cycle, Gestation, Offspring, and Uterine Contractions in Rats

Agomelatine is a pharmaceutical compound that functions as an agonist for melatonin receptors, with a particular affinity for the MT1 and MT2 receptor subtypes. Its mode of action is integral to the regulation of diverse physiological processes, encompassing the orchestration of circadian rhythms, sleep-wake cycles, and mood modulation. In the present study, we delve into the intricate interplay between agomelatine and the modulation of estrus cycles, gestation periods, offspring numbers, and uterine contractions, shedding light on their collective impact on reproductive physiology. Both in vivo and in vitro experiments were performed. Wistar Albino rats, divided into four groups: two non-pregnant groups (D1 and D2) and two pregnant groups (G1 and G2). The D1 and G1 groups served as control groups, while the D2 and G2 groups received chronic agomelatine administration (10 mg/kg). Uterine contractions were assessed in vitro using myometrial strips. Luzindole, a melatonin receptor antagonist, was employed to investigate the pathway mediating agomelatine's effects on uterine contractions. In in vivo studies, chronic agomelatine administration extended the diestrus phase (p<0.05) in non-pregnant rats, prolonged the gestational period (p<0.01), and increased the fetal count (p<0.01) in pregnant rats. Additionally, agomelatine reduced plasma oxytocin and prostoglandin-E levels (p<0.01) during pregnancy. In vitro experiments showed that agomelatine dose-dependently inhibited spontaneous and oxytocin-induced myometrial contractions. Luzindole (2 µM) reverse the agomelatine-induced inhibition of myometrial contractions. These findings suggest that agomelatine holds the potential to modulate diverse reproductive parameters during the gestational period, influencing estrus cycling, gestational progression, offspring development, and the orchestration of uterine contractions.

Effects of Achillea millefolium extract on spontaneous and oxytocin-induced isolated rat uterine contractions

Objective: Achillea millefolium (AM) is widely used in traditional medicine in a wide geography due to its effects on the female reproductive system. However, its effect on uterine smooth muscle contractions is unknown. Our study aims to investigate the effects of AM on spontaneous and oxytocin-induced isolated rat uterine contractions. 
 
 Method: Myometrial strips were obtained from 32 adult Wistar Albino rats. Contraction amplitudes and frequencies were recorded by isolated tissue bath system after either of the following: administration of only Krebs-Hanseleit (KBH) solution, administration of Achillea millefolium extract (AME); administration of KBH or AME after inducing contractions with oxytocin. The differences at the level of p

Assessing the Potency of the Novel Tocolytics 2-APB, Glycyl-H-1152, and HC-067047 in Pregnant Human Myometrium

The intracellular signaling pathways that regulate myometrial contractions can be targeted by drugs for tocolysis. The agents, 2-APB, glycyl-H-1152, and HC-067047, have been identified as inhibitors of uterine contractility and may have tocolytic potential. However, the contraction-blocking potency of these novel tocolytics was yet to be comprehensively assessed and compared to agents that have seen greater scrutiny, such as the phosphodiesterase inhibitors, aminophylline and rolipram, or the clinically used tocolytics, nifedipine and indomethacin. We determined the IC50 concentrations (inhibit 50% of baseline contractility) for 2-APB, glycyl-H-1152, HC-067047, aminophylline, rolipram, nifedipine, and indomethacin against spontaneous ex vivo contractions in pregnant human myometrium, and then compared their tocolytic potency. Myometrial strips obtained from term, not-in-labor women, were treated with cumulative concentrations of the contraction-blocking agents. Comprehensive dose–response curves were generated. The IC50 concentrations were 53 µM for 2-APB, 18.2 µM for glycyl-H-1152, 48 µM for HC-067047, 318.5 µM for aminophylline, 4.3 µM for rolipram, 10 nM for nifedipine, and 59.5 µM for indomethacin. A single treatment with each drug at the determined IC50 concentration was confirmed to reduce contraction performance (AUC) by approximately 50%. Of the three novel tocolytics examined, glycyl-H-1152 was the most potent inhibitor. However, of all the drugs examined, the overall order of contraction-blocking potency in decreasing order was nifedipine > rolipram > glycyl-H-1152 > HC-067047 > 2-APB > indomethacin > aminophylline. These data provide greater insight into the contraction-blocking properties of some novel tocolytics, with glycyl-H-1152, in particular, emerging as a potential novel tocolytic for preventing preterm birth.

Trivariate compound generalized poisson model for corticotropin-releasing hormone effects on human pregnant and nonpregnant

In this paper, we apply a new approach to uterine contractions to a large panel of human pregnant non pregnant myometrial strips, treated or not by corticotropin releasing hormone (CRH). This model is based on a fine analysis of the contraction curves.This analysis yield four mathematical parameters beta, theta, tau1 and tau2 related to excitability, duration of plateau phase, and time constants for relaxation describing respectively, the different portions of the contraction cycle. This leads to specific differences in spontaneous contractile activity between pregnant and nonpregnant states. In the present study, we compare nonpregant and pregnant women close to the term vitro CRH myometrial responsiveness, to describe uterine contractile activity during a parturition. We have a Trivariate compound generalized Poisson model for uterine contraction to a pregnant and nonpregnant myometrial strips, treated or not treated by corticotrophin- releasing hormone (CRH). Although, the biochemical signification of these results remains to be elucidated, we contribute to emphasize CRH action at the myometrical level. The results reveal highly significant differences between pregnant and nonpregnant myometrium as well as in their response to CRH.

Effect of metritis on in-vitro uterine contractility in cows during the puerperium

Aim of this study was to determine the effect of metritis on in-vitro uterine contractility. Uteri obtained from 16 euthanized Holstein-Friesian cows were divided into two groups depending on whether metritis was absent (M-, n = 6) or present (M+, n = 10). Four longitudinal and four circular myometrial strips of all uteri were incubated in an organ bath. Spontaneous contractility was recorded in five consecutive 30-minute periods (T1-T5). This was followed by stimulation of one longitudinal and one circular strip with increasing concentrations of oxytocin, prostaglandinF2α (PGF2α), and calcium chloride (each during four 30-minute periods [T6-T9]). Strips in group M+ had higher minimum amplitude (minA) values at T1 and higher minA, mean amplitude (meanA), and area under the curve (AUC) values at T2 than strips in group M- (P ≤ 0.05). In the M+ group, the maximum amplitude (maxA), meanA, and AUC values of circular strips were higher than those of longitudinal strips during spontaneous contractility (T1, T4, and T5; P ≤ 0.05). The minA, meanA, and AUC values for strips in group M+ were higher than those in group M- when exposed to the highest concentration of PGF2α (P ≤ 0.05). During stimulation with PGF2α (T9), longitudinal strips had higher maxA values than the circular strips in group M+ (P ≤ 0.05). Spontaneous and stimulated contractility were temporarily increased in uteri with metritis compared to healthy uteri. Both myometrial layers, especially in uteri with metritis, reacted differently during spontaneous contractility and to stimulation with PGF2α.

Dual effect of nifedipine on pregnant human myometrium contractility: Implication of TRPC1.

Nifedipine, an L‐type voltage‐gated Ca2+ channel (L‐VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions, but its efficacy is limited over time, and repeated or maintained nifedipine‐based tocolysis appears to be ineffective in preventing preterm birth. We aimed to understand why nifedipine has short‐lasting efficiency for the inhibition of uterine contractions. We used ex vivo term pregnant human myometrial strips treated with cumulative doses of nifedipine. We observed that nifedipine inhibited spontaneous myometrial contractions in tissues with high and regular spontaneous contractions. By contrast, nifedipine appeared to increase contractions in tissues with low and/or irregular spontaneous contractions. To investigate the molecular mechanisms activated by nifedipine in myometrial cells, we used the pregnant human myometrial cell line PHM1‐41 that does not express L‐VGCC. The in vitro measurement of intracellular Ca2+ showed that high doses of nifedipine induced an important intracellular Ca2+ entry in myometrial cells. The inhibition or downregulation of the genes encoding for store‐operated Ca2+ entry channels from the Orai and transient receptor potential‐canonical (TRPC) families in PHM1‐41 cells highlighted the implication of TRPC1 in nifedipine‐induced Ca2+ entry. In addition, the use of 2‐APB in combination with nifedipine on human myometrial strips tends to confirm that the pro‐contractile effect induced by nifedipine on myometrial tissues may involve the activation of TRPC channels.

An in vitro Study on the Contractility of Epileptic Myometrium and Effects of Antiepileptic Agents on Oxytocin-Induced Contractions of Myometrium Isolated from Absence Epileptic WAG/Rij Rats.

The aim of this study was to determine whether spontaneous and stimulated contractile activity of myometrium in epileptic rats is different from healthy ones, and whether antiepileptic drugs (AEDs) have any direct influence on myometrial contractility. Myometrial strips from nonpregnant and pregnant adult epileptic WAG/Rij and Wistar rats were suspended in organ bath containing physiological salt solution (37°C and pH 7.4, aerated with 95% oxygen-5% CO2), and isometric contractions were recorded. Effects of cumulative concentrations of selected AEDs including phenytoin, levetiracetam, and valproic acid alone and in combination on oxytocin-induced contractions was examined. Contractile parameters assessed included the area under curve, amplitude, and frequency of contractions, evaluated by 10-min periods. Data were analyzed using one-way analysis of variance and Tukey HSD test. Spontaneous myometrial contractility and responses to oxytocin showed species difference. Compared with that of control Wistar rats, spontaneous contractions of myometrium from nonpregnant epileptic WAG/Rij rats were significantly higher while being significantly lower in pregnant preparations. Upon stimulation with oxytocin, WAG/Rij myometrium showed significantly lower contractile response compared with preparations from healthy control Wistars (p < 0.01). Phenytoin and valproate caused concentration-dependent significant attenuation (p < 0.05) of spontaneous and oxytocin-induced contractions of myometrium from WAG/Rij and Wistar rats, both nonpregnant and pregnant. Myometrial smooth muscle from epileptic rats showed different spontaneous and oxytocin-induced contractility, and AEDs showed contractile modulatory actions, phenytoin being the most and levetiracetam the least effective. Although in vitro, our findings may be of clinical implications with regard to obstetric complications in epileptics and use of AEDs during pregnancy, and warrants further investigations.

Investigation of Effects of Gadolinium-Based Contrast Agents on Uterine Contractility Using Isolated Rat Myometrium.

Despite concerns about safety, gadolinium-based contrast agents (GBCAs) are still used for abdominal and pelvic imaging during pregnancy. Researchers have mainly focused on teratogenicity, while very little is known about their possible direct effects on uterine contractility, yet free gadolinium potentially impacts contractility through interaction with calcium channels. To investigate possible effects of selected GBCAs (namely gadoteridol, gadoversetamide, gadobutrol, gadoterate meglumine, and gadoxetic acid) on the contractility of rat myometrium. In vitro organ bath study. Myometria were isolated from adult (10-12 weeks old) Sprague Dawley rats, both pregnant (N=8) and nonpregnant (N=36). NA. Myometrial strips were suspended in tissue bath containing physiological saline and isometric contractions were recorded. GBCAs were added to the tissue bath cumulatively, and their effects on contractility parameters (quantified by amplitude, frequency, and area under contractility curve [AUC]) were evaluated by 10-minute intervals. Normality data, checked by Shapiro-Wilk test, were transformed by arcsine when needed. One- or two-way analysis of variance was performed, where appropriate, followed by Student-Newman-Keuls test. A P value of <0.05 was considered statistically significant. All of the assayed GBCAs elicited some alterations in the myometrial contractility in a concentration-dependent manner. Gadoterate meglumine, gadoxetic acid, and gadoversetamide caused a concentration-dependent significant attenuation in AUC (oxytocin-induced, from 100% during control period to 45.1 ± 9.0% (nonpregnant) and 59.9 ± 8.5% (pregnant), for 90 μM gadoterate meglumine; respectively), and frequency of the spontaneous and oxytocin-induced contractions. Gadobutrol and gadoteridol at highest dose significantly attenuated mean AUC and frequency of oxytocin-induced contractions of nonpregnant myometrium. Results from this in vitro study indicate that GBCAs elicit modulation of myometrial contractions at clinically relevant concentrations. These effects may account, at least partially, for the known potential side effects (rare cases of miscarriages and elective abortion) of these agents. 1 TECHNICAL EFFICACY: Stage 5.

Myometrial Responses to Beta-Adrenoceptor Antagonists in Gynecological Malignancies

Objective: The aim of the study was to determine the influence of beta-adrenoceptor (ADRB) antagonists on contractile activity of the nonpregnant human uterus in patients affected by gynecological malignancies. Design: This was a controlled and prospective ex vivo study. Setting: The work was conducted as a collaboration between 4 academic departments. Materials and Methods: Myometrial specimens were obtained from women undergoing hysterectomy for benign gynecological disorders (reference group; N = 15), and ovarian (N = 15), endometrial (N = 15), synchronous ovarian-endometrial (N = 3), and cervical cancer (N = 10). Contractions of myometrial strips in an organ bath before and after applications of ADRB antagonists (propranolol, bupranolol, SR 59230A, and butoxamine) were studied under isometric conditions. Results: Propranolol and bupranolol attenuated contractions in the endometrial and cervical cancer groups similar to that in the reference group (all p < 0.05), whereas opposite effects were observed in the ovarian and synchronous ovarian-endometrial cancer groups. SR 59230A and butoxamine significantly increased contractions in the ovarian cancer group (both p < 0.001). Limitations: These results require now to be placed into a firm clinical context. Conclusions: Our study indicates that ovarian cancer considerably alters contractile activity of the nonpregnant human uterus in response to ADRB antagonists. This suggests a pathogenetic role of beta-adrenergic pathways in this malignancy. Furthermore, propranolol and bupranolol substantially influence spontaneous uterine contractility.

Conflicting Nongenomic Effects of Progesterone in the Myometrium of Pregnant Rats.

Recently, it has been suggested that progesterone affects the contractile activity of pregnant myometrium via nongenomic pathways; therefore, we aimed to clarify whether progesterone causes and/or inhibits pregnant myometrial contractions via nongenomic pathways. Our in vitro experiments using myometrial strips obtained from rats at 20 days of gestation revealed that progesterone caused myometrial contractions in a concentration- and time-dependent manner at concentrations up to 5 × 10−7 M; however, this effect decreased at concentrations higher than 5 × 10−5 M. Similarly, progesterone enhanced oxytocin-induced contractions up to 5 × 10−7 M and inhibited contractions at concentrations higher than 5 × 10−5 M. Conversely, progesterone did not enhance high-KCl-induced contractions but inhibited contractions in a concentration- and time-dependent manner at concentrations higher than 5 × 10−7 M. We also found that RU486 did not affect progesterone-induced contractions or the progesterone-induced inhibition of high-KCl-induced contractions; however, progesterone-induced contractions were blocked by calcium-free phosphate saline solution, verapamil, and nifedipine. In addition, FPL64176, an activator of L-type voltage-dependent calcium channels, enhanced high-KCl-induced contractions and rescued the decrease in high-KCl-induced contractions caused by progesterone. Together, these results suggest that progesterone exerts conflicting nongenomic effects on the contractions of pregnant myometrium via putative L-type voltage-dependent calcium channels.

395 Dual effect of nifedipine on term pregnant human myometrium contractility

Nifedipine, L-type voltage-gated Ca2+ channels (L-VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions but its efficacy is limited overtime (48h). The aim of this study was to understand why nifedipine has short-lasting efficiency for inhibition of uterine contractions. Term pregnant human myometrial strips with different ex vivo contractile capacities [either regular (n=6) or irregular (n=5) spontaneous contractions] were mounted in an organ bath, and treated with cumulative doses of nifedipine. Tissue contractility in response to nifedipine was analyzed. Then, to investigate the molecular mechanisms activated by nifedipine in myometrial cells with irregular contractile capacity, we used the pregnant human myometrial cell line PHM1-41 that does express L-VGCC. Cells were transfected with siRNA for ORAI1, a non-voltage gated Ca2+ channel, and STIM1, the ORAI1 regulator. Both transfected and non-transfected cells were treated with different doses of nifedipine. Evolution of intracellular Ca2+ level was monitored with a Ca2+ indicator dye. Treatment of myometrial strips with cumulative doses of nifedipine inhibited spontaneous myometrial contraction in tissues with high contractility, even at low doses. On the contrary, nifedipine treatment appeared to increase contractions in tissues with low contractility (Fig1). In vitro, high doses of nifedipine (>10-6M) induced an important intracellular Ca2+ entry in myometrial cells (Fig2). Nifedipine-induced Ca2+ influx was not affected by knock-down of ORAI1 and STIM1 neither individually, nor together This study highlighted existence of a dual action of nifedipine. In addition to inhibit contraction in contracting myometrial tissue, we showed that nifedipine had an opposite pro-contractile effect in weak-contracting muscle. This effect could involve an activation of Ca2+ entry into myometrial cells, but this process does not seem to involve neither STIM1 nor ORAI1 Ca2+ channel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

668 Uterorelxant effect of adiponectin and omentin on In Vitro human myometrial contractility

The onset of parturition in pregnant women with obesity is frequently delayed. Without induction, these women are nearly twice as likely as normal-weight women to have prolonged pregnancies even after control for birth weight. Adipokines are cell signaling mediators secreted by the adipose tissue, many of which are now known to be closely linked to metabolic regulation including smooth muscle function. In this study, we aimed to investigate the role of adiponectin, and omentin on the in vitro functional contractility of isolated myometrial samples. Myometrial strips alongside the upper part of the low transverse uterine incisions were obtained at the time of the cesarean birth from 22 healthy, non-laboring, pregnant women, scheduled for elective cesarean birth at term. In isolated muscle tissue baths, myometrial strips were prepared under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative doses of adiponectin and omentin in the concentration range of 1 nmol/L to 10 umol/L. Control experiments were performed simultaneously. Both adiponectin and omentin exerted significant uterorelaxant effect ( reduction in amplitude and frequency of contractions) on the myometrial samples, in both spontaneous and oxytocin-induced contractions, in vitro. The inhibitory effect was in the region of 36 % for adiponectin, and 33 % for omentin in tissue baths with spontaneous contractions and in the region of 31% for adiponectin and 30% for omentin in tissue baths with oxytocin-induced contractions. In this study, we show the inhibitory role of adiponectin and omentin on human myometrial contractility, in vitro. These findings reiterate the physiological role of metabolic changes associated with maternal weight on myometrial contractility. The cellular mechanisms, and the potential clinical implications are not fully understood which could be addressed in future studies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

515 Repurposing of FDA-approved lead drugs as potentiators of uterine contractility in postpartum hemorrhage

Current medical options for management of postpartum hemorrhage preclude universal application due to their potential for adverse side effects. The objective of this study was to test the ex vivo efficacy of uterine-selective, FDA-approved drugs with no known cardiopulmonary toxicities repurposed for use at the human myometrial level. Lead drugs that selectively regulate human myometrial contractility were identified in prior studies using in vitro high throughput drug screening for calcium mobilization in myometrial cells. In this study, functional ex vivo isometric contractility was assessed via organ bath assays to determine uterotonic activity of mefloquine and pimavanserin on the human myometrial tissue. Human myometrial tissue was obtained at the time of cesarean delivery from non-laboring women meeting inclusion and exclusion criteria. Myometrial tissue strips (∼6-8 per biopsy) were submerged in organ baths (37°C, 95%O2, 5%CO2) containing Kreb’s Bicarbonate Solution and suspended under 3-gram isometric conditions. Tissues that displayed rhythmic spontaneous contractions were treated with cumulative concentrations (10pM – 0.1mM) of either mefloquine, pimavanserin or vehicle control (DMSO) at 20min intervals. Contractile activity was recorded and analyzed for frequency, amplitude, and area under the curve. Drug response relative to spontaneous contractile activity was analyzed, and non-linear regression models were used to generate concentration-response curves. Comparisons of fit were performed to determine if the non-linear curves of mefloquine and pimavanserin were significantly different (p<0.05) from DMSO. We performed 57 experiments in myometrial samples obtained from 12 women. There was no significant difference in the amplitude, frequency, or area under the curve between the vehicle control, mefloquine, and pimavanserin. Neither mefloquine nor pimavanserin induces ex vivo uterotonic activity in human myometrial tissue.

41In vitro human myometrial contractility in pregnancies complicated with obesity

The onset of parturition in pregnant women with obesity is frequently delayed. Without induction, these women are nearly twice as likely as normal-weight women to have prolonged pregnancies. Obesity has been shown to be an independent risk factor for cesarean birth, even after controlling for birth weight. It has been suggested that myometrial contractility is impaired in women with obesity. In this study we aimed to investigate whether contractility is truly reduced in women with obesity compared to their normal-weight counterparts utilizing an in vitro experiment model. Myometrial strips alongside the upper part of the low transverse uterine incisions were obtained at the time of the cesarean birth from 22 healthy, non-laboring, pregnant women, scheduled for elective cesarean birth at term. Strips were transferred to the laboratory and underwent dissection into tissue samples, each approximately 5 mm long, 2 mm wide, and 1 mm thick. In isolated muscle tissue baths, each tissue was attached to a stationary hook and the other end was attached to a force transducer and mounted for isometric recording under 2 gm of tension. Tissues were prepared under isometric conditions and underwent both spontaneous and oxytocin-induced contractions. Control experiments were performed simultaneously. Total of 11 participants with obesity and 11 normal-weight participants were enrolled. In both spontaneous and oxytocin-induced contractions, there was a significant reduction in amplitude of contractions between normal-weight and participants with obesity (P= 0.04, and P=0.049 in spontaneous and oxytocin-induced contractions respectively) and a significant reduction in frequency of contractions between groups (P= 0.01, and P=0.02 in spontaneous and oxytocin-induced contractions respectively). Obesity may impair the ability of the uterus to contract in labor which might contribute to the increased cesarean birth rate in women with obesity. This study reiterates the importance of preconception counseling on optimizing weight prior to pregnancy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)