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The synchronous contractions of the uterus in labour depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In the human myometrium, gap junctions are scarce in the non-pregnant uterus, but become abundant at term in preparation for labour. We have previously demonstrated that in the human myometrium at term, three different gap-junctional proteins are expressed, connexins 43, 45, and 40. These connexins are known to have distinctive functional capacities in in vitro expression systems but whether, in the human myometrium in vivo, they are co-assembled into the same gap junction or form different types of gap junction has previously been unclear. By applying triple immunogold labelling to sections of Lowicryl-embedded tissue for electron microscopy, together with complementary immunoconfocal microscopy, we demonstrate here that connexins 43, 45, and 40 are commonly present as mixtures within the same gap-junctional plaque. While all gap junctions contain connexin43, the relative signal for each connexin type varies between individual junctions. The presence within single gap-junctional plaques of three different connexins, each with the potential for conferring distinctive channel properties, suggests an inherent versatility for modulation of smooth muscle cell intercellular communication properties during human parturition., (Copyright 2001 Wiley-Liss, Inc.)

Objective: Our objective was to study the appearance and distribution of connexins 43 and 26 in various human myometrial cell cultures., Study Design: Scrape loading, Western blotting, and immunohistochemical techniques were applied to cultured cells derived from myometrial tissues obtained from nonpregnant and pregnant women (upper and lower uterine segments) and from leiomyomas (tumor and analogous myometrial tissues)., Results: Scrape loading revealed the presence of metabolic coupling in all tissues. Indirect immunohistochemical studies showed membrane localization of connexin 43 in all myometrial cultures. Western blots and indirect immunohistochemical studies showed the presence and localization of the connexin 26 protein and associated gap junctions in tissues from myomas and from nonpregnant and pregnant women except for those derived from the upper segment of the pregnant uterus., Conclusion: These results show that human myometrial cultures express various gap junction proteins and that there are regional differences in expression of connexins in tissues from pregnant women.

Objective: We sought to examine the change of cytosolic calcium concentration caused by prostaglandin F(2)(alpha) and RU 486 in cultured human myometrial cells., Study Design: Human myometrial cells obtained from 16 nonpregnant women were loaded with fura 2, and the intracellular cytosolic calcium concentrations were measured by the use of wavelength spectrophotofluorometry., Results: Application of prostaglandin F(2)(alpha) (2.8 micromol/L) caused an initial rapid rise in cytosolic calcium concentration followed by sustained cytosolic calcium oscillations at an average frequency of 0.43 +/- 0.04 min(-1) and an amplitude in the range of 296.82 +/- 27. 16 nmol/L. The oscillatory activity was not affected by increasing the concentration of prostaglandin F(2)(alpha) but varied by changing the concentration of extracellular cytosolic calcium concentration. The cytosolic calcium oscillations were suppressed by caffeine, 2,5-di-tert-butylhydroquinone, and lanthanum but not affected by ryanodine. Verapamil decreased the amplitude but not the frequency of oscillations. The progesterone antagonist RU 486 at a concentration of 10(-8) to 10(-5) mol/L had no significant effect on the basal intracellular cytosolic calcium. However, RU 486 (10(-5) mol/L) significantly increased the frequency but not the amplitude of intracellular cytosolic calcium oscillations induced by prostaglandin F(2)(alpha)., Conclusion: The results indicate that prostaglandin F(2)(alpha)-stimulated cytosolic calcium oscillations are mediated by an increase in both cytosolic calcium release from inositol 1,4,5-trisphosphate-sensitive cytosolic calcium stores and a cytosolic calcium influx from the extracellular space. Moreover, RU 486 seems to directly regulate prostaglandin F(2)(alpha)-induced intracellular cytosolic calcium in human myometrial cells.

Background: To investigate the mechanisms of oxytocin (OT) induced oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in cultured human myometrial cells., Methods: [Ca2+]i was measured in individual myometrial cells by dual wavelength spectrophotofluorometry using the fluorescent indicator fura-2. Myometrium was obtained at abdominal hysterectomy (n=8) and during cesarean section (n=7)., Results: OT (10-300 nM) typically induced [Ca2+]i oscillations with frequencies in the 0.6-0.8/min range. There were no obvious differences in the responses of cells taken from non-pregnant and term pregnant women. The frequency and amplitude of the oscillations were not significantly affected by OT concentrations up to 300 nM. The amplitude of the oscillations decreased in the presence of the voltage-dependent Ca2+ channel antagonist verapamil and gradually disappeared in Ca2+-free medium. The oscillations were further blocked by the inorganic Ca2+ antagonist La3+ and by the intracellular Ca2+-ATPase inhibitor 2.5-di-tert-butylhydroquinone (DTBHQ). Caffeine inhibited the OT-induced oscillations in a concentration-dependent manner. DTBHQ and high concentrations of OT made [Ca2+]i remarkably sensitive to changes in the external Ca2+ concentration., Conclusions: The results indicate that OT-induced [Ca2+]i oscillations in human myometrial cells are due to inositol 1,4,5-trisphosphate-mediated release of intracellular Ca2+ combined with capacitative as well as voltage-dependent influx of the ion.

Objective: To investigate the ability of human uterine myocytes to grow under anaerobic conditions for a prolonged time period., Methods: Cells were isolated from fundal myometrium and cultured until subconfluency. The cell type was confirmed by immunostaining for the smooth muscle cell-specific cytoskeletal proteins alpha-actin and desmin. Some cells were further cultured under aerobic conditions and others under anaerobic conditions. Cells were harvested after 0, 4 and 8 days in culture and analyzed for their content of adenylates., Results: Immunostaining revealed that all three preparations contained almost only smooth muscle cells. Energy charge of the myocytes was 0.88 on average at the beginning of the culture experiment. A moderate decrease was noted on day 4 for myocytes grown under both aerobic and anaerobic conditions and no further decrease was noted between days 4 and 8. Morphologically the cells retained their normal appearance and they seemed healthy for at least 8 days in culture under both aerobic and anaerobic conditions., Conclusions: The results of this study suggest that human myometrial cells can survive for an extended period of time under in vitro conditions regardless of oxygen availability for energy metabolism. This means that anaerobic energy metabolism is enough to sustain vital processes during that period of time., (Copyright 2000 S. Karger AG, Basel)

Glycogen, glucose, some nucleosides and purine base derivatives were determined in biopsies from 10 term pregnant women undergoing elective cesarean sections before labor. The concentrations were compared in the biopsies which were cut from isthmus and fundus of the uterus and m. rectus abdominis. M. rectus biopsies had 1.5 and 2 times higher concentration of glycogen than the isthmic and fundal uterine biopsies, respectively. The glucose concentration in the striated muscle was on the other hand only half that in the myometrium. The uterus displayed regional differences in concentrations of glycogen (higher in isthmus). A relatively low glycogen content in term pregnant uterine smooth muscle compared with striated muscle signifies the restricted energy demands of pregnant myometrium.

Objective: The effects of the uterotonic agent oxytocin on intercellular communication between term pregnant human myometrial cells before labor were studied to investigate its contribution to the synchronicity of uterine contractions., Study Design: The membrane potential and the input resistance of myometrial cells were measured with intracellular micropipettes, and dye-coupling assays were concomitantly performed while the tissues were perfused with three concentrations of oxytocin (10(-8) mol/L, 10(-7) mol/L, and 10(-6) mol/L). The results were compared with those obtained from tissues perfused with gap junctional uncoupling agent octanol and those with Tyrode's solution (control)., Results: Octanol increased input resistance and inhibited dye coupling but did not affect membrane potential compared with controls. Oxytocin depolarized membrane potential at all studied concentrations. The mean input resistance values and detectable dye propagation with oxytocin were similar to controls., Conclusion: Oxytocin does not contribute to gap junctional intercellular communication in term pregnant human myometrium before labor but may affect contractions by depolarizing membrane potential.

Objective: To study the relationship between the action of progesterone on term human myometrial contractile activity and cAMP alterations., Study Design: Myometrial tissues were obtained from 13 term pregnant women who underwent elective cesarean section. The contractile activity of muscle strips was recorded isometrically, and the level of cAMP in both superfusion media and experimental biopsies was measured by radioimmunoassay., Results: An increased cAMP release into the superfusion media and an increased accumulation of cAMP in myometrium were observed after the addition of progesterone (3, 15 and 150 microM). Simultaneously, the frequency and tonus of contractions were increased, while the amplitude and activity area of contractions were decreased., Conclusions: The data indicate that an increase in cAMP concentration plays a role in the action of progesterone on the regulation of term human myometrial contractile activity.

The powerful synchronous contractions of the uterus in labor depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In humans and other mammals, gap junctions are scarce in the myometrium of the non-pregnant uterus, but become abundant at term and/or with the onset of labor. Previous work has shown that the gap-junctional protein (connexin) expressed by human myometrial smooth muscle cells is connexin43, the same connexin type that predominates in cardiac muscle. Here we show that two further gap junctional proteins, connexin40 and connexin45, are expressed by the myometrial smooth muscle cells of the human uterus at term. Transcripts encoding the human isoforms of these connexins were demonstrated by Northern blot analysis, and immunoconfocal microscopy enabled precise localization of the corresponding proteins to punctate contact points (i.e., gap junctions) between interacting smooth muscle cells. Double labeling demonstrated that, while some fluorescent spots comprise only connexin43, both connexin40 and connexin45 predominantly colocalize to connexin43-positive fluorescent spots. Triple labeling revealed that where all three connexin types were expressed, they frequently localized to the same gap junction spot. As gap-junctional channels composed of different connexin types have been demonstrated in vitro to have different functional properties, multiple connexin expression may contribute to modulation of gap junction function in human myometrial smooth muscle cells in vivo.

Objective: Progesterone has been shown to have both stimulatory and inhibitory effects on term human myometrial contractile activity. The mechanisms involved in this action of progesterone are still poorly understood., Design: Myometrial tissues were obtained from the lower uterine segment at elective Caesarean section of 30 term pregnant women. The contractile activity of muscle strips was measured by a superfusion technique and protein synthesis evaluated by [3H]-leucine incorporation., Results: [3H]-leucine incorporation into term myometrial strips was not affected by progesterone (10 mumol/l), but was markedly reduced by the protein synthesis inhibitor anisomycin (P < 0.05). However, progesterone increased frequency and tonus of contractions and reduced the activity-area of contractions (P < 0.01). Anisomycin (100 mumol/l) did not change these effects or the spontaneous contractile activity. Removal or extracellular Ca2+ or addition of the L-type calcium channel blocker verapamil prevented the spontaneous as well as the progesterone-induced contractions, but had less pronounced effects on contractions initiated by oxytocin., Conclusion: The results indicate that the actions of progesterone on term myometrial contractile activity occur without protein synthesis and that increased Ca2+ influx or decreased outward transport of Ca2+ may play a possible role)

The purpose of the study was to investigate by in vitro and in vivo methods the phosphocreatine (PCr)/ ATP ratio as an expression of the energy metabolic state of human myometrium in comparison with striated skeletal muscle. The contents of PCr and adenylates in biopsies of uterine smooth muscle and m. rectus abdominis from seven term pregnant women were determined in vitro and compared with results obtained in vivo by phosphorus magnetic resonance spectroscopy (31P-MRS) in the uterus and m. gastrocnemius of eight non-pregnant women. The PCr/ATP ratio in the striated skeletal muscle was about three times higher than that of the myometrium. The results of the in vitro biopsy part of the study and the in vivo 31P-MRS part conformed with each other. In the biopsies both PCr and ATP concentrations were significantly lower in the myometrium than in the rectus muscle, but the difference for PCr was more pronounced, accounting for the significantly lower PCr/ATP ratio in the uterine smooth muscle. The energy metabolic pattern of uterine smooth muscle differs from that of striated skeletal muscle regarding the contents of high-energy phosphocompounds and the PCr/ATP ratio. This in vivo finding is the first report on human smooth muscle using 31P-MRS.

The aim of this study was to quantify gap junction expression in the human myometrium in relation to progesterone and oestradiol concentrations, and to establish whether oxytocin-resistant dystocia is due to an abnormality in gap junction expression. Three groups of patients were investigated: (i) before labour (at term), (ii) normal labour and (iii) oxytocin-resistant dystocia (eight patients per group). For each patient, the concentrations of oestradiol and progesterone in maternal blood and in myometrial tissue were measured, and the number and area of immunostained connexin43 gap junctions per unit volume of tissue determined by quantitative analysis of digital images obtained by confocal microscopy. No significant difference in connexin43 gap junction content was observed between the three patient groups. When all groups were pooled, there was a significant positive correlation (P < 0.05) between the quantity of immunolabelled gap junctions and the oestradiol:progesterone ratio, but there was no significant difference in this correlation between the groups. Gap junction immunolabelling was not correlated with the progesterone or oestradiol concentration in the maternal blood or the myometrium. These data suggest that in human myometrium: (i) dystocia is not due to a reduced level of immunodetectable connexin43 gap junctions, (ii) onset of labour is not associated with a sudden increase in immunodetectable gap junction protein and (iii) gap junctions can be expressed in the presence of high progesterone concentrations.

Objective: To study the effects of progesterone and oxytocin on contractile activity related to the intracellular elemental composition of human pregnant myometrium., Study Design: Myometrial strips were mounted in tissue baths and superfused with plain buffer, oxytocin or progesterone. Progesterone was initially added to the tissue bath or at the onset of spontaneous contraction of the strips. The myometrial contractile activities were recorded isometrically and the results were analyzed by a specific computer program. The effects of oxytocin and progesterone on the intracellular elemental composition were studied by X-ray microanalysis in an electron microscope., Results: The contractile activity in terms of frequency and tonus of the muscle strips was increased by oxytocin and progesterone. This increase was more pronounced if progesterone was added to the bath at the start of the experiment. After 120 min of incubation with oxytocin and progesterone the total intracellular concentration of calcium was significantly higher (P < 0.0001 and P < 0.002, respectively) compared to buffer., Conclusions: (i) Oxytocin increases total intracellular calcium concentration concomitant with an increase in uterine contractile activity. (ii) Progesterone increases the myometrial tone and frequency of contractions, simultaneously with an increase in total intracellular calcium.

The appearance of gap junctions (GJs) between myometrial smooth muscle cells is one of the major events associated with the onset of labor. We have employed dye-coupling and electrical-current injection techniques to study the mechanisms by which steroid hormones regulate GJs in term pregnant myometrium of women before labor. Progesterone (P4) did not alter the input resistance (Ro) of the tissues when added to Tyrode's solution, which was used as control treatment. Octanol, the putative gap junctional uncoupling agent, increased the Ro of the cells compared to the control and P4-treated groups. The membrane potential (Em) did not differ between these groups. However, when P4 was applied after the tissue was perfused with estradiol (E2), the results changed dramatically: the Em hyperpolarized, and the Ro increased. Octanol increased the Ro in E2-treated tissues, but did not affect the Em. Consecutive application of E2, octanol, E2, and P4 resulted in rapid changes in the Ro of the cells. Dye-coupling was mostly detected between cells from controls and E2-treated tissues. These results indicate that P4 exerts its effects in the presence of E2 and that P4 has rapid effects on the intercellular communication between human myometrial cells.

Previous studies by electron microscopy and laser confocal microscopy of immunohistochemically stained sections have shown that during pregnancy the extent of gap junction formation in human myometrium is low, but that an increase occurs in the active stages of labour despite a high concentration of progesterone in maternal blood. The present investigation focused on the effect of in vitro exposure of isolated myometrial tissue to progesterone, oestradiol and oxytocin, on the number of gap junction plaques in human myometrium at term. Myometrial biopsies were obtained at term from 13 pregnant women who had an elective caesarean section in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer and buffer containing 0.5, 5.0 micrograms mL-1 of progesterone, and 0.1 micrograms mL-1 of oestradiol. The muscle biopsies were trimmed under a stereo microscope into strips along the bundles of smooth muscle cells and mounted in tissue baths, superfused with Hepes buffer supplemented with glucose (0.01 mM); subsequently the strips were exposed to buffer containing different concentrations of progesterone, oestradiol and oxytocin. Incubation of the muscle strips for 180 min resulted in a significant decrease of the number of gap junctions (P < 0.01). Neither oestradiol or oxytocin, alone or in combination, had a significant effect on the maintenance of the number of gap junctions. The progesterone concentration of 5.0 micrograms mL-1, combined with oxytocin, and with or without oestradiol has a significantly positive effect on the number of gap junction plaques in strips of human myometrium at term (P < 0.05 vs. buffer alone). The high concentration of progesterone in the superfusion medium during in vitro experiments may be responsible for the maintenance of high numbers of gap junction complexes in term human myometrium. This finding is of interest in the light of findings of persisting high progesterone levels in maternal blood during labour.

The aim of this study was to measure oxytocin receptor concentration in myometrial tissue from term pregnant women with normal and dysfunctional labor and to relate this concentration to the progress of labor and to the levels of estradiol and progesterone in the same myometrium. Myometrial biopsies were obtained from 50 term pregnant women undergoing cesarean section. The patients were categorized as follows: not in labor, normal labor, successful oxytocin-augmented labor, and oxytocin-resistant labor. Specific binding of [3H]oxytocin to high-affinity sites in membrane preparations from myometrial tissues was determined. Estradiol and progesterone were assayed using tritiated steroids with a sensitive radioimmunoassay technique. Oxytocin receptor density was significantly lower in oxytocin-resistant labor compared to successful oxytocin-augmentated labor (P < 0.04) and to spontaneously active normal labor (P < 0.02). Oxytocin receptor concentration was also significantly lower in non-labor patients compared to normal spontaneous labor (P < 0.01), and successful oxytocin-augmented labor (P < 0.02). There was a positive relationship between the progress of cervical dilatation (cm/h) and oxytocin receptor density in the myometrium (r = 0.408, P < 0.025). The concentration of progesterone and estradiol in the pregnant myometrium did not differ in patients with different types of labor or with the state of uterine contractile activity. Our results suggest that individual myometrial sensitivity is an important determinant of the response to administered oxytocin in humans. Furthermore, myometrial oxytocin receptor expression in vivo seems not be related to ovarian steroid concentration in the myometrium. The low oxytocin receptor density in oxytocin-resistant dystocia needs further investigation.

The concentrations of 24 amino acids and four other related compounds were determined in extracts of biopsy specimens from myometrium and musculus rectus abdominis of 10 healthy term pregnant women during elective cesarean sections. The total free amino acid pool did not differ significantly between the two muscle types. There was a myometrial abundance of the two anionic amino acids glutamate and aspartate and the two aromatic amino acids tyrosine and phenylalanine, but most neutral and cationic amino acids in striated skeletal muscle tissue were found in excess of those in uterine muscle tissue.

The purpose of this study was to investigate the elemental composition of myometrial cells from term parturient women with normal and dysfunctional labour. Myometrial biopsies were obtained from forty-four term pregnant women undergoing Cesarean section. The patients were categorized according to uterine activity as follows: before labour, normal labour, labour successfully augmented by oxytocin, oxytocin-resistant labour, and labour arrested by terbutaline. X-ray microanalysis of freeze-dried sections (16 mu m thick) of the myometrium was carried out. An increase in intracellular phosphorus level (p < .01) was noted in the normal labour group compared to before labour. In patients with normal labour, higher phosphorus (p < .009) and potassium (p < .005) were found compared to oxytocin resistant labour. Patients with oxytocin resistant labour had lower intracellular potassium (p < .0006) and phosphorus (p < .02), and higher chloride (p < .05) and sodium (p < .03) compared to levels found in patients who responded to oxytocin treatment. In dysfunctional (oxytocin-resistant) labour the ion distribution in the myometrial cells might be disturbed. The reduced level of potassium and phosphorus together with the high sodium and chloride levels found in patients with oxytocin resistant labour may be connected to an impairment in sodium-potassium pump and muscle dysfunction, clinically diagnosed as dystocia.

Objective: To study the energy metabolite pattern in term pregnant myometrium and to compare it with that of a striated skeletal muscle., Study Design: Metabolites of carbohydrates, triglycerides and nucleotides were determined in biopsies from myometrium and rectus abdominis muscle from ten healthy term pregnant women before onset of labour. The metabolite concentrations in the two types of muscle were compared by two tailed Student's t-test for paired values and correlations were calculated by Pearsson's correlation test., Results: Comparison of the two muscle types revealed a higher concentration of glucose and a higher lactate/pyruvate ratio in the myometrium but a lower concentration of triglyceride metabolites. Adenosine was found in 36-fold higher concentration in the uterine compared with the rectus muscle and an inverse relationship between adenosine and glucose concentrations was observed in the myometrium., Conclusion: The results indicated that the pregnant uterine smooth muscle utilized glucose as the principal nutritive metabolite rather than lipids and that the anaerobic pathway of the glucose metabolism was more active in the myometrium compared with the striated rectus muscle. Also, it is suggested that glucose has a critical role as the principal fuel for ATP formation and the involvement of the adenylate kinase and 5'-nucleotidase reactions in any event of glucose shortage.

Objective: To study the energy economy of human myometrium in vitro., Study Design: Myometric biopsies were kept for 10 min or 4 h in a buffer solution at +4 degrees C before preparation into 'early' and 'late' strips, respectively. The strips were perifused at +37 degrees C for different periods of time. Adenylates, energy charge (EC), adenosine and NAD+ were determined in individual strips., Results: Mean EC and ATP values were low, the 'late' strips displaying 5% and 20% lower values than the 'early' strips, respectively. During perifusion the EC increased slightly, ADP decreased significantly and the strips began to contract. A correlation fitting a multiplicative function was evident between EC and NAD+ (r = 0.89, P < 0.001)., Conclusion: Human myometrium, being operative at an unusually low EC level, maintained its energy economizing and contractile functions in vitro for 4 h, displaying a tight relationship between EC and NAD+, suggestive of a compromised energy balance.

Background and Methods: Some structures in the human myometrium, namely; caveolae, extracellular space and gap junctions were quantified in tissues from nonpregnant women, women who were not in labor, and women in spontaneous labor or oxytocin-induced labor, in order to reveal morphologic differences that would contribute to explaining the events leading to labor. Transmission electron micrographs taken from tissues from twenty-four pregnant and ten nonpregnant women were studied., Results: Differences were observed in cell growth which was accompanied by enlargement of the extracellular space, and in gap junctions. Gap junctions were demonstrated in nonpregnant women, and even in one postmenopausal woman. Their frequency and size increased gradually between groups of pregnant women in the order of non-labor, oxytocin-induced labor and spontaneous labor. The increase in size of the junctions was less than the increase in number., Conclusions: Gap junctions are most frequently found in human myometrial cells in spontaneous labor, which strongly suggests a role for these structures in the termination of pregnancy. The effect of oxytocin on the appearance of gap junctions was negligible; hence, the contribution of oxytocin to establish myometrial contractile synchronicity is thought to be minor. Caveolae are probably not relevant for the initiation of labor as their distribution did not differ between the groups.

Term pregnant human myometrial cells in whole mounts were microinjected by pressure with the fluorescent probes Lucifer Yellow and carboxyfluorescein. Tissues obtained from acute and elective sections displayed weak dye-coupling when injected with Lucifer Yellow. Injection of carboxyfluorescein into cells from the elective sections resulted in a more extensive dye-coupling than that observed with Lucifer Yellow. These results indicate that term pregnant human myometrial cells are metabolically coupled before labor and carboxyfluorescein is superior to Lucifer Yellow in detecting the coupling.

In human myometrium, at various stages of labour and in correlation with the concentration of progesterone and estradiol in maternal blood the formation of gap junctions has been described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated human myometrium at term to Hepes buffer, on the number of gap junction plaques. Biopsies of myometrium were obtained from 5 pregnant women at term: they had an elective caesarean operation in the 37th or 40th week of pregnancy. The biopsies were immersed immediately in Hepes buffer, trimmed under a stereo microscope into small strips and kept in warm (37 degrees C) oxygenated Hepes buffer supplemented with glucose (0.01 mM). Then some strips of myometrium were incubated at 37 degrees C for 10, 20, 40, 55, and 180 min. The number of gap junction plaques were counted: it decreased in strips of myometrium after 10, 20, 40, 55, and 180 min of in vitro experiment (P < 0.01 vs control). In some of our experiments, the decrease in quantity of gap junction plaques was very dramatic and far below 50% of their numbers found in control specimens but never reached the zero value.

Objective: To investigate the interactions of progesterone, estradiol (E2), and oxytocin in regulating the contractile activity of term human myometrium in vitro., Methods: Myometrial tissues from 12 term pregnant women were obtained from the lower uterine segment at elective cesarean and immediately immersed in Hepes buffer with and without progesterone and progesterone plus E2. Oxytocin was freshly dissolved in solutions when the superfusion was started. Muscle biopsies were dissected along the bundles of smooth muscle under a stereomicroscope, and the preparations were mounted in six tissue baths superfused with different hormonal solutions. The isometric tension was recorded during the experimental time., Results: During the 2-hour contraction period, increased frequency (P < .01 for each) and decreased activity area (P < .01 for each) and duration of contractions (P < .01 for each) resulted from superfusion of muscle strips with progesterone plus oxytocin, progesterone plus E2, and a combination of progesterone, E2, and oxytocin, compared to that found with buffer alone. The higher concentration of progesterone (5 micrograms/mL) plus oxytocin and progesterone plus E2 elevated the tonus (P < .05 and P < .01), whereas progesterone in combination with E2 and oxytocin decreased it (P < .05). The lower concentration of progesterone (0.5 microgram/mL) plus E2, or both in conjunction with oxytocin, had no significant effect on tonus as compared with buffer., Conclusion: The results suggest that progesterone and E2 have a direct effect on uterine excitability and a regulating influence on the oxytocin sensitivity of term human myometrium.

In the human myometrium, the formation of gap junctions in the various stages of labor and, in correlation with the concentration of progesterone in the maternal blood, has not been described adequately. The accepted hypothesis that raised concentrations of progesterone in maternal blood in animals prevent the formation of gap junctions was scrutinized in human myometrium. Myometrial tissue was examined by electron microscopy for the presence of gap junctions and analyzed morphometrically in 17 women who had an elective or emergency cesarean operation at the 38th or 39th week of pregnancy. The concentrations of progesterone and estradiol in the maternal and cord blood was measured by means of radioimmunoassay. The frequency and area of gap junctions varied in relation to the presence or absence of labor. In the myometrium of women in labor, gap junctions were most frequent (5.2/1000 microns of cell membrane), their area largest (0.32 microns 2/1000 microns of cell membrane) and their fractional area greatest (0.20%). When the women were not in genuine labor or when contractions were weak, the percentage fractional areas of gap junctions were low (0.020-0.090) and the gap junctions very small. In the pre- and active phases of labor, the concentration of progesterone in maternal blood was significantly higher (p < 0.025 and 0.03 respectively) than in women who were not in labor. Hence, despite high concentrations of progesterone in the maternal and cord blood at term, the myometrium presented numerous gap junctions of relatively large area.

Direct intracellular microinjection of a fluorescent dye (Lucifer Yellow) was performed on ex-situ muscle strips from term pregnant women not in labor. The aim was to characterize the intracellular distribution of LY to obtain criteria for an intracellular injection in whole mounts and ultimately to study the gap junctional communication between myometrial cells in those tissues. Fifteen injections performed on biopsies from ten cases showed a well-bordered fusiform shape and were considered to be intracellular. The intercellular spread of the dye into an adjacent cell was observed in three injections (three cases). The average cell dimension was 284 +/- 95 microns for length and 5 +/- 1.5 microns for width (n = 17). The intracellular injection was confirmed by light microscopy of cross sections. It is concluded that limited coupling exists between myometrial cells of women prior to labor.

Gap junctions are thought to play an essential role in the control of parturition. In this study we attempted to synthesise three short peptides which match the portion of the amino acid sequence of the connexin43, protein predicted from the nucleotide sequence of cDNA clone and to produce an anti-peptide antibody which would detect gap junction protein in human myometrium. Three peptides were synthesized: (1) RKEEKLNKKEEE, (2) EJKKFKYGJEEH and (3) KNRVKGRSDPYH. Antibodies against these peptides were raised in rabbits, screened for reactivity and specificity using dot blot assays and used for immunocytochemical staining of heart and myometrial tissues.

We determined the concentrations of adenosine and some of its catabolic products in biopsy specimens from predetermined loci of human myometrium under different functional conditions to compare uterine muscle with rectus abdominis muscle from the same individuals. In order to achieve a good resolution in the separation of nucleosides and purine bases, a preseparation procedure was developed prior to analysis of these compounds on high performance liquid chromatography. Adenosine occurred in a nearly 70-fold higher concentration in smooth uterine muscle in comparison with striated skeletal muscle. Similarly, myometrial inosine and hypoxanthine were 7- and 2.4-times in excess over the rectus muscle, whereas xanthine was scarcely and rather evenly represented in the two types of muscles. The uterine content of adenosine and inosine was distinctly higher in pregnant women compared to non-pregnant ones. A regional difference existed for adenosine, with 3.3 times higher concentration in fundus uteri compared to the isthmic part. A reverse pattern was observed for hypoxanthine and inosine, being 2-3 times more frequent in the isthmic part. The orthophosphate concentration was not stoichiometrically related to the adenosine concentration in a simple way, being 2-3 times lower in uterine muscle compared to the skeletal muscle. A significant correlation existed between uterine contents of AMP and adenosine and similarly, significant inverse correlations were apparent between uterine ATP and ADP contents and energy charge on one hand and adenosine content on the other.

Gap junctions are of importance in the coordinated contraction of uterine smooth muscle during labor. There are indications that in the human myometrium, gap junctions are much more numerous in the period immediately surrounding parturition. The presence and the functional state of gap junctions in human myometrium was studied by electron microscopical morphometry and by studying the distribution of an injected fluorescent dye. Electron microscopy showed that the number and size of gap junctions was significantly higher during the active phase of labor than during the pre-labor phase. Pressure injection of Lucifer Yellow into myometrial cells in vitro showed only weak coupling in the pre-labor stage. These data support the notion that coupling between myometrial cells develops in immediate association with parturition.

The concept that the progesterone-estrogen ratio determines gap junction abundance in myometrium has developed from studies in laboratory animals. In the humans, however, gap junctions form in abundance at term despite a high progesterone-estrogen ratio. We have therefore examined the relationship between contractile activity, progesterone and estrogen levels and gap junction abundance in human myometrium at term. Myometrial tissue obtained from women who had an elective or emergency Cesarean operation at the 38th or 39th week of pregnancy was immunohistochemically examined using a polyclonal antiserum raised against a synthetic peptide matching part of the sequence of connexin43, the principal component of myometrial gap junctions. The concentration of progesterone and estradiol in the maternal blood was measured by means of radio-immunoassay. The number of gap junctions was estimated by counting of punctate staining patterns obtained in myometrial tissue as viewed by laser confocal microscopy and calculated per 1 microns 3 of tissue. The number of gap junctions measured was found to be related to the presence or absence of labor. In the myometrium of women in labor (active phase), the number of gap junctions was highest (45.25 +/- 35). When the myometrium was not contracting (non-labor) and when contractions were weak (pre-labor) the number of gap junctions was low (15.16 +/- 10-5.75 +/- 5). In the pre-and active phase of labor, the concentration of progesterone in maternal blood was significantly higher (P < 0.025 and 0.03, respectively) than in women who were not in labor. The present study shows the potential of using optical section series obtained by laser confocal microscopy for the quantification and mapping of the three-dimensional distribution of gap junctions in human myometrium at term.

Twelve pregnant women at term with unfavorable cervices (less than or equal to 5 points according to Bishop score) were given prostaglandin E2 for cervical priming and labor induction. Prostaglandin E2 was given in the following manner: 0.5 mg in gel strictly intracervically (n = 4), extraamniotically (n = 4), or prostaglandin E2 (4 mg) in gel vaginally (n = 4). The myometrial activity was registered over a period of 30 minutes before and at least 3 hours after gel application by means of an extraamniotic microtransducer catheter. After both extraamniotic and vaginal gel application, myometrial activity was significantly increased compared with intracervical application. All the women had favorable cervical states 6 hours after treatment and were delivered vaginally within 24 hours. All infants were delivered in good condition with 1-minute Apgar scores greater than 7. From these results we conclude that proper intracervical prostaglandin E2 gel application, in contrast to extraamniotic or vaginal application, induces cervical ripening without significant myometrial activity. Because careful intracervical application appears to avoid or minimize the risks of myometrial hyperstimulation, this technique should be considered particularly in women with unfavorable cervices and delicate fetuses.

Using monoclonal antibody assay techniques estrogen receptors (ER) and progesterone receptors (PgR) were quantitated in female pelvic floor muscles, urogenital ligaments and uterus (myometrium). Receptors were detected in all these structures. In addition qualitative assessment showed distinct nuclear staining in the same structures. ER and PgR were not found in the musculus rectus abdominis with either method. The findings provide a rationale for estrogen treatment in women with urogenital disorders.

In 7 primigravidae admitted for first trimester abortion by dilatation and evacuation, 0.5 mg PGE2 in viscous gel (5 patients) or placebo gel (2 patients) was applied intracervically 6 hours prior to the operation. Throughout the treatment period intrauterine pressure was recorded. Application of placebo gel induced no cervical ripening or myometrial activation. In all patients receiving active gel, a marked improvement of the cervical state was induced by the treatment. In three cases, this priming occurred in parallel to minimal changes in myometrial activity, without regular uterine contractions. In two patients, marked uterine activation was registered due to partly extraamniotic application. It is suggested, that the PGE2-gel has a direct effect on the cervical tissues. Further, the risk of partially applying the gel in the extraamniotic space, thus stimulating the myometrium, depends on the gel volume relative to the dimensions of the cervical canal and the application technique.

A micro-transducer catheter was tested in vitro and in vivo for recording of myometrial activity in the non-pregnant human uterus and was also compared with conventional fluid-filled, open end catheters previously used for recording intra-uterine pressures. The micro-transducer catheter had a frequency response far above that of fluid-filled, open end catheters. It was easily introduced through the uterine cervix and was never obstructed. Provided that the sensory surface of the micro-transducer was kept from direct contact with the uterine wall by some special arrangement it gave recordings of the intra-uterine pressure, that were practically identical with those obtained by an open end catheter.

The effects of nicardipine, a calcium-entry blocker which also has a potent phosphodiesterase inhibitory action, were investigated on isolated human term-pregnant and nonpregnant myometrium, and compared with those of nifedipine. Both drugs relaxed pregnant and nonpregnant myometrial preparations contracted by potassium (127 mM), and also reduced or abolished contractions occurring spontaneously, or induced by prostaglandin F2 alpha, oxytocin and vasopressin. However, the effect of nicardipine had a slower onset of action than that of nifedipine, and the drug was significantly more potent than nifedipine is at least as effective as nifedipine. If the differences between the drugs can be reproduced also in vivo, nicardipine offers an interesting alternative to nifedipine for inhibition of undesired uterine activity.

Uterine hypercontractility is considered to be an important factor in primary dysmenorrhea. A survey is given on possible mechanisms controlling the cytoplasmic concentration of free calcium and thereby contractile activity in the myometrial smooth muscle cell. Probably acting by different modes of action, inhibitors of prostaglandin synthesis, calcium antagonists and beta 2 stimulators have all been shown to reduce myometrial activity and relieve dysmenorrheic pain. The possibility of achieving further uterine relaxation after initial treatment with prostaglandin inhibitors by adding a calcium antagonist such as nifedipine is discussed. It is also suggested that when utilizing a reliable pressure recording technique in the evaluation of dysmenorrheic patients, the pronounced myometrial relaxation obtained by such combined therapy may be of diagnostic value.

Myometrial tissue was obtained from non-pregnant women subjected to hysterectomy because of various gynaecological disorders, and from women undergoing caesarean section. Strip preparations were dissected and isometric tension was recorded. Nifedipine (2.9 X 10(-8)--2.9 X 10(-6)M) inhibited spontaneous contractile activity, mainly by reducing the amplitude of contraction in both non-pregnant and pregnant myometrium. The drug also inhibited potassium induced contractions in a concentration dependent manner. This effect seemed to be more pronounced in pregnant than in non-pregnant tissue. In preparations of pregnant human myometrium, normally polarized or potassium depolarized, oxytocin induced a contractile activity that was effectively inhibited by nifedipine. Nifedipine also relaxed contractions induced by vasopressin in isolated non-pregnant myometrium. It is concluded that the relaxant effect of nifedipine on isolated pregnant and non-pregnant human myometrium can be explained by inhibition of calcium influx. The results thus support the view, that calcium influx is an important step in the initiation of contractile activity in human uterine smooth muscle.

In twelve women, in whom labor was induced by oxytocin infusion, intra-uterine pressure (IUP) was recorded by micro-transducers, and simultaneously the diameter of the uterine cavity was estimated by means of ultrasound. Myometrial tension was calculated according to the law of Laplace. In the ten women who were delivered within eight hours after the start of induction, myometrial tension was clearly higher than in the two in whom induction failed. No technical problems were encountered during the registrations, which in some cases lasted for more than five hours. It is suggested that with the described technique, a more accurate determination of myometrial activity is possible than with the generally used methods for IUP recording. The technique might facilitate evaluation of drug effects on the uterus during labor, and improve the IUP-part of intrapartum clinical monitors.