Pregnant Myometrium Research Articles

Correlation of the Levels of Oxytocin Receptor and the Enzymes for Prostaglandin Synthesis to NF-�eB Activation in Human Pregnant Myometrium at Term

The switch from pregnancy to delivery involves various changes in expression of uterine activation proteins (UAPs). Increasing body of evidence indicates that human labor is an inflammatory event which results in the activation of nuclear factor kappa B (NF-κB) in uterus. Our aim was to analyze the correlation between the level of activated NF-κB and the expression of UAPs including oxytocin receptor (OTR), cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), and prostaglandin F synthase (PGFS) in pregnant human myometrium at term. The myometrial tissues were obtained from pregnant women who were in labor (TL) or not in labor (TNL) at term. Western blotting was used to determine the level of activated NF-κB and the expression of OTR, COX-2, mPGES-1 and PGFS in pregnant myometrium. The expression of OTR, COX-2, mPGES-1 and PGFS was significantly up-regulated in TL group compared with TNL group (P ＜ 0.01). The level of activated NF-κB was significantly higher in TL tissues than that in TNL biopsies (P ＜ 0.01). Significant positive correlations were found between the level of activated NF-κB and the expression of OTR (P＜0.05), COX-2 (P＜0.05), mPGES-1 (P＜0.01) or PGFS (P＜0.01). Upregulation of UAPs such as OTR, COX-2, mPGES and PGFS is correlated to the activation of NF-κB in pregnant human myometrium at term. Our data support the hypothesis that inflammation leads to uterus activation for labor.

203: Maternal body mass index and spontaneous contractile activity of pregnant human myometrium in vitro

203: Maternal body mass index and spontaneous contractile activity of pregnant human myometrium in vitro

202: Human myometrial in vitro effects of pharmacological agents used in the clinical management of postpartum hemorrhage

ObjectivePostpartum hemorrhage (PPH) is a leading cause of maternal mortality in the developed and under-developed world, and uterine atony is the major contributing factor. Clinical guidelines for drug treatment of PPH vary internationally, and there are minimal comparative pharmacological data for the various uterotonic compounds used. The aim of this study was to evaluate a range of contractile parameters in human pregnant myometrium, for the uterotonic agents used clinically in the management of atonic PPH.Study DesignMyometrial biopsies were obtained from 19 patient donors undergoing elective cesarean delivery at term. The effects of the uterotonic agents oxytocin, carbetocin, ergometrine, carboprost, syntometrine and misoprostol were investigated in 146 myometrial strips. The potency (expressed as pEC50) and maximal response values were obtained, and compared, using both maximal amplitude and mean contractile force as indices of contraction. Single EC50 concentrations of the agents were administered and both force and contraction peak parameters were compared during a 15 minute exposure.ResultsThe most important difference between the agents was in their ability to increase the mean contractile force, with oxytocin superior to all agents except syntometrine. In single dose experiments mean contractile force was the parameter that separated the agents. Oxytocin was not statistically different from carboprost or syntometrine but was superior to all other agents. There was a wide difference in potencies using both measures of contractility, with oxytocin and carbetocin being most potent. There were no significant differences in the peak amplitude of response between agents, except for misoprostol, which was inactive.Conclusion ObjectivePostpartum hemorrhage (PPH) is a leading cause of maternal mortality in the developed and under-developed world, and uterine atony is the major contributing factor. Clinical guidelines for drug treatment of PPH vary internationally, and there are minimal comparative pharmacological data for the various uterotonic compounds used. The aim of this study was to evaluate a range of contractile parameters in human pregnant myometrium, for the uterotonic agents used clinically in the management of atonic PPH. Postpartum hemorrhage (PPH) is a leading cause of maternal mortality in the developed and under-developed world, and uterine atony is the major contributing factor. Clinical guidelines for drug treatment of PPH vary internationally, and there are minimal comparative pharmacological data for the various uterotonic compounds used. The aim of this study was to evaluate a range of contractile parameters in human pregnant myometrium, for the uterotonic agents used clinically in the management of atonic PPH. Study DesignMyometrial biopsies were obtained from 19 patient donors undergoing elective cesarean delivery at term. The effects of the uterotonic agents oxytocin, carbetocin, ergometrine, carboprost, syntometrine and misoprostol were investigated in 146 myometrial strips. The potency (expressed as pEC50) and maximal response values were obtained, and compared, using both maximal amplitude and mean contractile force as indices of contraction. Single EC50 concentrations of the agents were administered and both force and contraction peak parameters were compared during a 15 minute exposure. Myometrial biopsies were obtained from 19 patient donors undergoing elective cesarean delivery at term. The effects of the uterotonic agents oxytocin, carbetocin, ergometrine, carboprost, syntometrine and misoprostol were investigated in 146 myometrial strips. The potency (expressed as pEC50) and maximal response values were obtained, and compared, using both maximal amplitude and mean contractile force as indices of contraction. Single EC50 concentrations of the agents were administered and both force and contraction peak parameters were compared during a 15 minute exposure. ResultsThe most important difference between the agents was in their ability to increase the mean contractile force, with oxytocin superior to all agents except syntometrine. In single dose experiments mean contractile force was the parameter that separated the agents. Oxytocin was not statistically different from carboprost or syntometrine but was superior to all other agents. There was a wide difference in potencies using both measures of contractility, with oxytocin and carbetocin being most potent. There were no significant differences in the peak amplitude of response between agents, except for misoprostol, which was inactive. The most important difference between the agents was in their ability to increase the mean contractile force, with oxytocin superior to all agents except syntometrine. In single dose experiments mean contractile force was the parameter that separated the agents. Oxytocin was not statistically different from carboprost or syntometrine but was superior to all other agents. There was a wide difference in potencies using both measures of contractility, with oxytocin and carbetocin being most potent. There were no significant differences in the peak amplitude of response between agents, except for misoprostol, which was inactive. Conclusion

387: The mitochondrion and uterine myocyte senescence in human pregnant myometrium: implications for labor and maternal age

387: The mitochondrion and uterine myocyte senescence in human pregnant myometrium: implications for labor and maternal age

Cu(II) complexation does not affect oxytocin action on pregnant human myometrium in vitro.

Copper may influence the in vivo and in vitro uterine activity. Recent evidence shows that cupric ions can easily form complexes with oligopeptides like oxytocin (OXT). The high complex stability in vitro suggests a possibility of complex formation in vivo. In vitro isometric contractions were recorded in uterine tissues from pregnant women undergoing caesarean sections and the effect of OXT and the Cu-OXT complex on isolated human pregnant myometrium was investigated. In the concentration range from 10(-14) to 10(-6)M of OXT alone, pre-formed Cu-OXT complex, and OXT following sample preincubation with Cu(II) salt, nosignificant differences were observed for the following parameters of pregnant uterine smooth muscle contraction: the area under the curve, frequency and amplitude of contraction. The binding of Cu(2+) ions does not abolish the ability of OXT to interact with oxytocin receptors and stimulate myometrial contraction in vitro.

METHODOLOGICAL ASPECTS OF IMMUNOHISTOCHEMICAL STAINING FOR von WILLEBRAND FACTOR IN TISSUES OF UTERO-PLACENTAL AREA AND MYOMETRIUM IN PREGNANCY

Introduction. There are different methods of morphological examination of uterine-placental area (UPA) and myometrium. The most effective of them is biopsy of tissues, taken within cesarean section. Biopsy specimens are applicable for the whole spectrum of modern morphological methods, including immunohistochemical staining. Development of the procedure for von Willebrand factor (vWF) detection in the myometrium of pregnant uterus is very important, as it was never used for these tissues. Aim and objectives. This research was aimed to assess methodologically the prospects and practical value of immunohistochemical staining for vWF in uterine-placental area and myometrium in pregnancy. Material and methods. UPA and myometrial biopsy specimens were taken by own original method. Tissues were fixed in 10% neutral buffered formalin solution for 22-24 hours, followed by ethanol dehydration and paraffin embedding. Multiple paraffin sections at the 5 mkm thickness were used for three methods: 1) immunohistochemical vWF staining with an antigen thermal exposure (DAKO); 2) differentiated histochemical fibrin and collagen staining by N.Z.Slinchenko; 3) hematoxylin and eosin staining. Digital images were analyzed with a special computer program ImageJ for histological studies (1.48v, free license, W. Rasband, National Institute of Health, USA, 2015). The evaluation of optical color density (ranging from 0 to 1 ) was based on logarithmic transformations of brightness values (in 0to255 skale). We also calculated the arithmetic mean and its error For optical density (computer program PAST 3.06, free license, O.Hammer, 2015). Results and discussion. Pronounced positive staining for vWF most often was observed in endothelial cells of formed blood vessels. Distribution of vWF staining intensity in endothelial cells is promising for studying endothelial dysfunction in different types of vessels of various localization (myometrium or UPA). The procedure outlined here has shown the ability to identify desquamated endothelial cells in the vascular lumen, whereas precise morphological verification of these cells is usually not impossible due to immediate anoikis processes in them (a type of apoptosis). In histological sections we have detected some groups of cells, forming new blood vessels in myometrial stroma and particularly in UPA. Thus, immunohistochemical staining for vWF in myometrium and UPA allows to estimate not only the angiogenesis (formation of new blood vessels from pre-existing ones), but also vasculogenesis (formation of new blood vessels when there are no pre-existing ones). The study has shown that given method of staining for vWF might reveal intravascular blood clots in myometrium and UPA on early stage of thrombosis, especially under the slow blood flow. Besides, this technique is more precise, compared to classic fibrin tests. There also was interesting finding of vWF UPA fibrinoid. This is to confirm, that immunohistochemical staining for vWF can detect as late, as early phases of UPA fibrynoid formation (even before fibrin formation). The lust is an ultimate condition for fibrinoid changes of uterus walls and for rearrangement of uterine spiral arteries, which are the most favorable for the normal fetus development. Conclusion. Immunohistochemical staining for vWF in utero-placental area and myometrium of pregnant women methodologically allows to evaluate the processes of angiogenesis (restructuring of existing vessels); vasculogenesis (formation of new blood vessels); endothelial dysfunction in different types of blood vessels; the processes of early and late thrombosis; processes of gestational reorganization of spiral arteries and, finally, formation of fibrynoid in uterine-placental area.

Maternal Age and Contractility of Human Myometrium in Pregnancy.

There is controversy as to whether maternal age exerts an influence on the contractility of human myometrium in pregnancy. The aim of this study was to examine a series of functional contractile parameters of human myometrium in vitro, over a broad range of maternal ages. Myometrial tissue specimens were obtained at cesarean delivery from 32 women with maternal ages ranging from 28 to 52 years. Using in vitro recordings, a number of contractile parameters including maximal amplitude, mean contractile force, time to maximal amplitude, maximum rate of rise, and occurrence of simple and complex (biphasic and multiphasic) contractions were examined for spontaneous and induced contractile activity. The relationship between maternal age and individual parameters was evaluated using linear regression analysis. For all contractile parameters examined, for both spontaneous and induced contractions, no significant correlation was observed with maternal age between 28 and 52 years. The mean maximum amplitude values for spontaneous and oxytocin-induced contractions were 23 ± 3 and 43 ± 5 mN, respectively. The mean contractile forces for spontaneous and oxytocin-induced contractions were 1.5 ± 0.2 and 6.5 ± 0.9 mN, respectively. There was no variation in the proportion of biphasic or multiphasic contractions with maternal age. These results indicate there is no significant functional impairment of uterine contractility and no lack in responsiveness of myometrium in vitro, in the older mother. These findings do not support the concept that there may be a biological basis for dysfunctional labor or increased cesarean delivery rates in older parturients.

Effects of d- and l-limonene on the pregnant rat myometrium in vitro.

AimTo study the effects of d- and l-limonene on pregnant rat myometrial contractility in vitro, and investigate how these effects are modified by other agents. D- and l-limonene (10−13-10−8 M) caused myometrial contraction in a dose-dependent manner.MethodsContractions of uterine rings from 22-day-pregnant rats were measured in an organ bath in the presence of d- or l-limonene (10−13-10−8 M) and nifedipine (10−8 M), tetraethyl-ammonium (10−3 M), theophylline (10−5 M), or paxilline (10−5 M). Uterine cyclic adenosine monophosphate (cAMP) level was detected by enzyme immunoassay. Oxidative damage was induced by methylglyoxal (3 × 10−2 M) and the alteration was measured via noradrenaline (1 × 10−9 to 3 × 10−5 M) -induced contractions.ResultsPre-treatment with nifedipine (10−8 M), tetraethylammonium (10−3 M), and theophylline (10−5 M) attenuated the contracting effect of d- and l-limonene, while in the presence of paxilline (10−5 M) d- and l-limonene were ineffective. The two enantiomers decreased the myometrial cAMP level, but after paxilline pretreatment the cAMP level was not altered compared with the control value. Additionally, l-limonene (10−6 M) diminished consequences of oxidative damage caused by methylglyoxal (3 × 10−2 M) on contractility, whereas d-limonene was ineffective.ConclusionOur findings suggest that l-limonene has an antioxidant effect and that both d-and l-limonene cause myometrial contraction through activation of the A2A receptor and opening of the voltage-gated Ca2+ channel. It is possible that limonene-containing products increase the pregnant uterus contractility and their use should be avoided during pregnancy.

Physiological increases in lactate inhibit intracellular calcium transients, acidify myocytes and decrease force in term pregnant rat myometrium.

Lactate is increased in myometrial capillary blood from women in slow or non-progressive labour (dystocia), suggesting that it is detrimental to uterine contractions. There are, however, no studies of the effect of lactate on the myometrium. We therefore investigated its effects and mechanism of action on myometrial strips from term pregnant rats. The effects on spontaneous and oxytocin-induced contractility in response to sodium lactate and other weak acids (1-20 mM) were studied. In some experiments, simultaneous force and intracellular Ca(2+) or pH (pH(i)) were measured with Indo-1 or Carboxy-SNARF, respectively. Statistical differences were tested using non-parametric tests. Lactate significantly decreased spontaneous contractility with an EC50 of 3.9 mM. Propionate, butyrate and pyruvate also reduced contractions with similar potency. The effects of lactate were reduced in the presence of oxytocin but remained significant. Lactate decreased pH(i) and nulling the decrease in pH(i) abolished its effects. We also show that lactate inhibited Ca(2+) transients, with these changes mirroring those produced on force. If Ca(2+) entry was enhanced by depolarization (high KCl) or applying the Ca(2+) channel agonist, Bay K 4644, the effects of lactate were abolished. Taken together, these data show that lactate in the physiological range potently decreases myometrial contractility as a result of its inhibition of Ca(2+) transients, which can be attributed to the induced acidification. The present study suggests that the accumulation of extracellular lactate will reduce myometrial contractions and could therefore contribute to labour dystocia.

CD34 and CD49f Double-Positive and Lineage Marker-Negative Cells Isolated from Human Myometrium Exhibit Stem Cell-Like Properties Involved in Pregnancy-Induced Uterine Remodeling.

Repeated and dramatic pregnancy-induced uterine enlargement and remodeling throughout reproductive life suggests the existence of uterine smooth muscle stem/progenitor cells. The aim of this study was to isolate and characterize stem/progenitor-like cells from human myometrium through identification of specific surface markers. We here identify CD49f and CD34 as markers to permit selection of the stem/progenitor cell-like population from human myometrium and show that human CD45– CD31– glycophorin A– and CD49f+ CD34+ myometrial cells exhibit stem cell-like properties. These include side population phenotypes, an undifferentiated status, high colony-forming ability, multilineage differentiation into smooth muscle cells, osteoblasts, adipocytes, and chondrocytes, and in vivo myometrial tissue reconstitution following xenotransplantation. Furthermore, CD45– CD31– glycophorin A– and CD49f+ CD34+ myometrial cells proliferate under hypoxic conditions in vitro and, compared with the untreated nonpregnant myometrium, show greater expansion in the estrogen-treated nonpregnant myometrium and further in the pregnant myometrium in mice upon xenotransplantation. These results suggest that the newly identified myometrial stem/progenitor-like cells influenced by hypoxia and sex steroids may participate in pregnancy-induced uterine enlargement and remodeling, providing novel insights into human myometrial physiology.

HSPB8 and the Cochaperone BAG3 Are Highly Expressed During the Synthetic Phase of Rat Myometrium Programming During Pregnancy1

The small heat shock protein (HSP) B family of proteins are a group of molecular chaperones that enable tissues to adapt to changes in their local environments during differentiation, stress, or disease conditions. The objective of this research was to characterize the expression of HSPB8 and its cochaperone Bcl2-associated athanogene 3 (BAG3) in nonpregnant (NP) and pregnant rat myometrium during myometrial programming. Rat myometrium was collected from NP and pregnant rats as well as 1 day postpartum (PP) and samples prepared for immunoblot and immunofluorescence analysis. Immunoblot analysis determined that HSPB8 protein expression was significantly elevated at Day (D) 15, D17, and D19 compared to expression at NP and D6, while BAG3 expression was significantly elevated at D15 compared to NP, and D17 compared to NP, D6, D23, and PP time points (P < 0.05). In situ, HSPB8 and BAG3 were predominantly localized to myometrial cells throughout pregnancy, with intense cytoplasmic HSPB8 and BAG3 detection on D15 and D17 in both longitudinal and circular muscle layers. Immunoblot analysis of HSPB8 and BAG3 protein expression in myometrium from unilateral pregnancies also revealed that expression of both proteins was significantly increased at D15 in gravid compared to nongravid horns. Thus, HSPB8 and BAG3 are highly expressed during the synthetic phase of myometrial differentiation marked by initiation of uterine distension and myometrial hypertrophy. HSPB8 and BAG3 could be regulators of the protein quality control required for this process.

Exogenous oxytocin modulates human myometrial microRNAs

MicroRNAs (miRNAs) play a modulatory role in pathways that lead to labor onset, although oxytocin is known to modulate gene expression within the myometrium. We aimed to identify miRNAs whose expression is regulated by oxytocin in pregnant human myometrium. Myometrial miRNA expression profiles were compared between samples collected from women at term before the onset of labor (no labor; n = 8) and after labor onset after early exogenous oxytocin treatment (n = 8). Multivariate modelling was used to assess differences in miRNA profiles. Biologic validation was undertaken on 3 independent patient cohorts (no labor, n = 10; labor induced with oxytocin, n = 8; and spontaneous labor with no oxytocin treatment, n = 10). In vitro studies that used primary myocytes were undertaken to assess target miRNA expression after oxytocin treatment. Target genes of candidate miRNAs were identified in silico and cross-referenced with genes that are known to be associated with labor or expressed in myometrium. In total, 1309 miRNAs were analyzed by microarray, of which 494 were detected in human myometrium. Multivariate modeling identified 12 target miRNAs the differential expression of which was most responsible for the observed separation of the 2 patient populations in the primary discovery cohorts. Biologic validation in the independent secondary sample cohorts showed that oxytocin independently regulated 5 miRNAs (hsa-miR-146b-3p, hsa-miR-196b-3p, hsa-miR-223-3p, hsa-miR-873-5p, and hsa-miR-876-5p). Additionally, hsa-miR-146b-3p was increased both in labor that was induced with oxytocin and in myometrium from spontaneous labor with no oxytocin treatment compared with no labor samples. Four of the validated miRNAs (hsa-miR-146a-5p, hsa-miR-146b-3p, hsa-miR-196b-3p, and hsa-miR-876-5p) were expressed in primary human myocytes; oxytocin treatment of these cells replicated the directional changes that were observed in vivo. Oxytocin alters the expression of a unique set of myometrial miRNAs. These results suggest a further role for oxytocin as a signaling molecule that is involved in the regulation of gene expression during parturition.

Effects of the polyphenol resveratrol on contractility of human term pregnant myometrium.

The ideal agent for prevention and treatment of uterine abnormal contractility has not been found. The polyphenol resveratrol possesses a wide spectrum of pharmacologic properties, but its influence on the contractility of human myometrium is not defined. The present study evaluated the effect of resveratrol on the oxytocin-induced contractions of human term pregnant myometrium in vitro and the contribution of different K(+) channels to resveratrol action. Resveratrol induced a concentration-dependent relaxation of myometrium contractions (pD2 value and maximal responses were 4.52 and 82.25%, respectively). Glibenclamide, a selective blocker of ATP-sensitive (KATP), iberiotoxin, a selective blockers of big-calcium sensitive (BK(Ca)) and 4-aminopiridine, a non-selective blocker of voltage-sensitive (Kv) channels induced a significant shift to the right of the concentration-response curves of resveratrol. Inhibition achieved by 0.1 mM resveratrol was insensitive to all K(+) channel blockers. A K(+) channel opener, pinacidil, inhibited oxytocin-induced contractions of pregnant myometrium with comparable potency and efficacy to resveratrol (pD2 values and maximal relaxation were 4.52 and 83.67%, respectively). Based on K(+) channel opener/blocker affinities, it appears that the inhibitory response of resveratrol involves different myometrial K(+) channels. When applied in high concentrations, resveratrol has an additional K(+)-channel-independent mechanism(s) of action. Furthermore, immunohistochemistry staining and western blot analyses detected the presence and distribution of KATP, BK(Ca) and Kv channel proteins in pregnant myometrium.

Effects of nociceptin and nocistatin on uterine contraction.

The presence and effects of nociceptin (N/OFQ) and nocistatin (NST) in the central nervous system have been reasonably well described, but less data are available on their peripheral functions. Besides their presence in several peripheral organs (white blood cells, airway, liver, skin, vascular and intestinal smooth muscles, ovary, and testis), they have been found in the pregnant myometrium in both rat and human. The level of their precursor prepronociceptin is elevated in the preterm human myometrium as compared with full-term samples, whereas it gradually increases toward term in the pregnant rat uterus. Both N/OFQ and NST inhibit myometrial contractions, an effect which can be enhanced by naloxone and blocked by Ca²⁺-dependent K⁺ channel (BK(Ca)) inhibitors. Both compounds increase the myometrial cAMP level which may be responsible for the activation of this channel and subsequent intracellular hyperpolarization. NST releases calcitonin gene-related peptide from the sensory nerve ends, which explains its cAMP-elevating effect. In contrast with the nervous system, where they behave as antagonists, N/OFQ and NST are able to potentiate the uterine-relaxing effect of each other in both rat and human tissues. Further studies are required to clarify the roles of N/OFQ and NST in the regulation of the myometrial contractions and the perception of pain during delivery.

5-HT3 receptor antagonists do not alter spontaneous contraction of pregnant myometrium in vitro

Background5-HT3 receptor antagonists are effective antiemetics for perioperative use. However, their effects on myometrial contractility remain unknown. We examined whether three different 5-HT3 receptor antagonists could affect the contraction of human myometrium. MethodsSamples of human myometrium were taken from parturients undergoing elective cesarean delivery. Effects of ondansetron, granisetron and tropisetron (over a range of 1–104ng/mL) on spontaneous contraction (ratios of amplitude, interval, and duration of the contraction) were examined and compared to saline controls (n=6 for each agent). ResultsNone of the three 5-HT3 receptor antagonists significantly affected myometrial contraction. Conclusion5-HT3 receptor antagonists do not affect the contraction of myometrial strips isolated from term pregnant women.

Transcriptomic analysis of the myometrium during peri-implantation period and luteolysis--the study on the pig model.

In pigs, implantation begins with the attachment of embryos to the endometrium. As the process is regulated by the expression of numerous genes, endometrial transcriptomic profiles have been extensively studied in early gravid pigs. However, the myometrium, a secretory tissue, should not be neglected, as it can also participate in the regulation of implantation in early pregnant pigs. To clarify this issue, the transcriptomic profile of the porcine myometrium during the peri-implantation period (i.e. on days 15 to 16 of pregnancy) was compared with the profile observed during luteolysis (i.e. on days 15 to 16 of the oestrous cycle) with an Agilent’s Porcine (V2) Two-Colour Gene Expression Microarray 4 × 44 (Agilent, USA). Analysis of the microarray data revealed that of 526 unique, accurately annotated genes, the expression of 271 unique genes was upregulated, while the expression of 255 genes was downregulated in pregnant versus cyclic myometrium. The in-depth data analysis revealed differential expression of genes encoding for factors involved in immunomodulation, tissue growth and differentiation, and prostaglandin and steroid biosynthesis and action. Moreover, the comparison of the obtained data on the myometrial transcriptome with our previously published results on the endometrial transcriptome allowed us to determine substantial differences in the regulatory function of both tissues. The new insights into the function of the myometrium of early pregnant pigs obtained here are in agreement with our previous results that suggest that this tissue plays an important role in providing optimal conditions for developing embryos. Therefore, the importance of the myometrium as an active embryo signal-responsive tissue during early pregnancy cannot be underestimated.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-014-0401-4) contains supplementary material, which is available to authorized users.

Isolated human uterine telocytes: immunocytochemistry and electrophysiology of T-type calcium channels.

Recently, telocytes (TCs) were described as a new cell type in the interstitial space of many organs, including myometrium. TCs are cells with very long, distinctive extensions named telopodes (Tps). It is suggested that TCs play a major role in intercellular signaling, as well as in morphogenesis, especially in morphogenetic bioelectrical signaling. However, TC plasma membrane is yet unexplored regarding the presence and activity of ion channels and pumps. Here, we used a combination of in vitro immunofluorescence and patch-clamp technique to characterize T-type calcium channels in TCs. Myometrial TCs were identified in cell culture (non-pregnant and pregnant myometrium) as cells having very long Tps and which were positive for CD34 and platelet-derived growth factor receptor-α. Immunofluorescence analysis of the subfamily of T-type (transient) calcium channels CaV3.1 and CaV3.2 presence revealed the expression of these ion channels on the cell body and Tps of non-pregnant and pregnant myometrium TCs. The expression in TCs from the non-pregnant myometrium is less intense, being confined to the cell body for CaV3.2, while CaV3.1 was expressed both on the cell body and in Tps. Moreover, the presence of T-type calcium channels in TCs from non-pregnant myometrium is also confirmed by applying brief ramp depolarization protocols. In conclusion, our results show that T-type calcium channels are present in TCs from human myometrium and could participate in the generation of endogenous bioelectric signals responsible for the regulation of the surrounding cell behavior, during pregnancy and labor.

Long-term type 1 diabetes alters the deposition of collagens and proteoglycans in the early pregnant myometrium of mice.

We have previously shown that long-term type 1 diabetes affects the structural organization, contractile apparatus and extracellular matrix (ECM) of the myometrium during early pregnancy in mice. This study aimed to identify which myometrial ECM components are affected by diabetes, including fibril-forming collagen types I, III and V, as well as proteoglycans, decorin, lumican, fibromodulin and biglycan. Alloxan-induced type 1 diabetic female mice were divided into subgroups D1 and D2, formed by females that bred 90-100 and 100-110 days after diabetes induction, respectively. The deposition of ECM components in the myometrium was evaluated by immunohistochemistry/immunofluorescence. The subgroup D1 showed decreased deposition of collagen types I and III in the external muscle layer (EML) and decreased collagen types III and V in the internal muscle layer (IML). Collagen types I and III were decreased in both muscle layers of the subgroup D2. In addition, increased deposition of collagen types I and III and lumican as well as decreased collagen type V were observed in the connective tissue between muscle layers of D2. Lumican was decreased in the EML of the subgroups D1 and D2. Fibromodulin was repressed in the IML and EML of both D1 and D2. In contrast, decorin deposition diminished only in muscle layers of D2. No changes were noticed for biglycan. Subgroups D1 and D2 showed distinct stages of progression of diabetic complications in the myometrium, characterized by both common and specific sets of changes in the ECM composition.

The effects of methyl palmitate, a putative regulator from perivascular fat, on the contractility of pregnant human myometrium

AimsMethyl palmitate is thought to cause relaxation in vascular smooth muscle by opening voltage-activated potassium channels. We have tested the hypothesis that methyl palmitate, a putative regulator from perivascular fat, is an inhibitor of the contractility of human pregnant myometrium and that its effects might partially explain the higher incidence of dysfunctional labor in obese women compared to those with normal body mass indices. Main methodsStrips of myometrium obtained with informed consent from women undergoing elective cesarean section at term were mounted in organ baths. Strips stimulated with oxytocin (1nM) or KCl (30mM) were exposed to cumulatively increasing concentrations of methyl palmitate up to 10μM. Similar strips were exposed to cumulative addition of the potassium channel blockers 4-aminopyridine and tetraethylammonium. The contractility of the strips was monitored and analyzed using conventional methods. Key findingsMethyl palmitate failed to inhibit oxytocin- or KCl-induced contractions over the concentration range tested. In fact, it exerted a slight excitatory effect in the presence of KCl, though not in the presence of oxytocin. The contractility of naïve strips was unaltered by exposure to 1μM methyl palmitate. Both 4-aminopyridine and tetraethylammonium produced concentration-dependent contractions of human pregnant myometrium providing pharmacological evidence for the presence of voltage-activated potassium channels in this preparation. SignificanceOur findings do not support the hypothesis that methyl palmitate is an inhibitor of human pregnant myometrial contractility. Alternate hypotheses must be pursued to explain the higher incidence of dysfunctional labor in obese women.

The role of progesterone receptor isoforms in the pregnant myometrium

Preterm birth complicates 10-12% of pregnancies worldwide with significant economic and healthcare burdens. Progesterone is necessary to establish and maintain pregnancy. Humans undergo a functional progesterone withdrawal for initiation of labor, possibly due to changes in the ratio progesterone receptor isoforms PRA and PRB, as there is an increase in the PRA/PRB ratio at the time of labor. Our goal is to determine the role of these PR isoforms in the maintenance of pregnancy by altering the isoform ratio in the mouse myometrium.