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The human uterus has no pacemaker or motor innervation, yet develops rhythmic, powerful contractions that increase intrauterine pressure to dilate the cervix and force the fetus through the pelvis. To achieve the synchronous contractions required for labor, the muscle cells of the uterus act as independent oscillators that become increasingly coupled by gap junctions toward the end of pregnancy. The oscillations are facilitated by changes in resting membrane potential that occur as pregnancy progresses. Reductions of potassium channels in the myocyte membranes in late pregnancy prolong myocyte action potentials, further facilitating transmission of signals and recruitment of neighboring myocytes. Late in pregnancy prostaglandin production increases leading to increased myocyte excitability. Also late in pregnancy myocyte actin polymerizes allowing actin-myosin interactions that generate force, following myocyte depolarization, calcium entry, and activation of myosin kinase. Labor occurs as a consequence of the combination of increased myocyte to myocyte connectivity, increased depolarizations that last longer, and activated intracellular contractile machinery. During labor the synchronous contractions of muscle cells raise intrauterine pressure to dilate the cervix in a process distinct from peristalsis. The synchronous contractions occur in a progressively larger region of the uterine wall. As the size of the region increases with increasing connectivity, the contraction of that larger area leads to an increase in intrauterine pressure. The resulting increased wall tension causes myocyte depolarization in other parts of the uterus, generating widespread synchronous activity and increased force as more linked regions are recruited into the contraction. The emergent behavior of the uterus has parallels in the behavior of crowds at soccer matches that sing together without a conductor. This contrasts with the behavior of the heart where sequential contractions are regulated by a pacemaker in a similar way to the actions of a conductor and an orchestra., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The pregnant uterus is unique because of the dramatic functional changes that occur in the peripartum period. To promote the concept that we have a relatively poor understanding of the physiology of parturition, we will posit 10 facts that are so obvious and so clearly accepted as facts that they probably are not even facts at all. (1) The laboring uterus undergoes peristalsis to dilate the cervix, deliver the fetus, and expel the placenta. (2) The human uterus is composed of longitudinal and circular layers of smooth muscle. (3) The functional cells of the uterus are the myocytes, which are a homogeneous cell type responsible for the generation of contraction forces, passage of action potentials, and control of contractility. (4) The phasic contractions of the uterus are typical for visceral smooth muscle. (5) The primary, and perhaps only, role of gap junctions is to allow passage of action potentials through the tissue. (6) Action potential propagation as the mechanism for global communication (over many centimeters throughout the uterus) is sufficient to recruit all regions and all myocytes of the uterus. (7) Slow waves pace the contractions of human myometrium. (8) Calcium-activated potassium channels are responsible for repolarization of the membrane potential that terminates each contraction. (9) Chloride channels are not important in uterine electrophysiology. (10) With enough computing power, it would be straightforward to build a closed model of human labor, given our current understanding of the components of myometrium. This manuscript discusses each point to stimulate questions for future investigation.

The objective of this study was to develop a three-dimensional culture system for the study of human myometrial physiology. Primary cell lines were initiated from human myometrium obtained at the time of term cesarean delivery. After several passages, cells were seeded onto a polyglactin-910 (Vicryl) mesh and maintained in culture. After several days in culture, each mesh was transferred to another culture dish and suspended to avoid contact with the plastic of the dish. Time-lapse videomicroscopy was used to observe cell proliferation and three-dimensional (3-D) fill of the pores of the mesh. Membrane potentials of the cells of this 3-D tissue were measured with a conventional microelectrode. Confocal microscopy was used to assess 3-D morphology. In some experiments, cells were seeded onto two layers of mesh and then cultured as described above. In this two-mesh experiment, force was measured by anchoring one mesh and pulling on the other, using a micrometer-driven strain gauge. In the single-mesh experiment, cells grew into and filled the pores of the mesh by repetitive proliferation, retraction, and proliferation. A confluent, 3-D tissue was obtained within 10 to 14 days of the initial seeding of the mesh. The average membrane potential of the cells within the single mesh was -35 +/- 6 mV. Confocal microscopy demonstrated tissue thickness of 9 to 40 microm (one to eight cells) within the pores of the mesh. In the two-mesh experiment, 2 to 3 weeks in culture yielded confluent 3-D tissues, in which myocytes not only filled the pores of each mesh, but also bridged between the two meshes. The bridging myocytes were able to maintain a tension of 5 g/cm(2) before separation of the two meshes, and coordinated contractions of 40 to 200 cells were observed. We conclude that cultured human myocytes proliferate and form 3-D tissues when supported by Vicryl scaffolding. Tissue grown in 3-D may provide a model system that is sufficient to probe the physiology of cell-to-cell interactions in myometrium.

Objective: The purpose of this study was to characterize the oxytocin-induced desensitization of oxytocin-stimulated rises of intracellular calcium in cultured human myocytes., Study Design: Culture lines were begun from biopsy specimens of myometrium that had been obtained from women who underwent low transverse cesarean deliveries. Fluorescence changes of calcium green-1 were used to demonstrate the rises of intracellular free calcium. Cells were exposed to 10 nmol/L oxytocin for 1 to 6 hours before the experimentation, allowed to rest for 10 minutes, and then tested for the fluorescence increases that resulted from exposure to 10 nmol/L oxytocin and micromol/L prostaglandin F(2)(alpha). Subpopulations were defined as type 1 (responded to both oxytocin and prostaglandin F(2)(alpha)), type 2 (responded only to oxytocin), type 3 (responded only to prostaglandin F(2)(alpha)), or type 4 (responded to neither). The distribution of the subpopulations of cells was assessed by the determination of the response of every cell in every experimental run., Results: Pretreatment with oxytocin resulted in a decrease in the percentage of cells that responded to subsequent oxytocin exposure. The decrease was dependent on the duration of oxytocin exposure and was well fit with the Boltzmann sigmoid function. The duration of oxytocin exposure that yielded half-inactivation was 4.2 hours. Without oxytocin pretreatment, the distribution of subpopulations were 37.0% +/- 18.0% (type 1), 23.1% +/- 11.5% (type 2), 12.6% +/- 8.0% (type 3), and 27.3% +/- 22.9% (type 4). After 6 hours of oxytocin pretreatment, the percentage of type 1 and type 2 cells decreased to 2.4% +/- 3.8% and 2.6% +/- 2.4%, respectively, although the percentage of type 3 and type 4 cells increased to 20.4% +/- 18.9% and 74.6% +/- 22.1%, respectively., Conclusion: Oxytocin-induced desensitization of myocytes to oxytocin stimulation occurred over a clinically relevant time frame (4.2 hours). Continued responsiveness of the cells to prostaglandin F(2)(alpha) stimulation after 6 hours of oxytocin pretreatment indicated that postreceptor signaling pathways were maintained, which indicates that the oxytocin receptor likely is involved in the mechanism of myocyte desensitization to oxytocin stimulation.

Cultured human myocytes exhibit intercellular calcium waves that travel farther than 100 microm ('far waves'). This work investigates the mechanism of far wave propagation. Culture lines were initiated from myometrial biopsies of term pregnant women. Calcium green-1 was used as a fluorescence probe for intracellular free calcium. Serial imaging was performed at a frame rate of 0.83 frames/s. Intercellular calcium waves were mechanically initiated by atraumatically applying small drops of mineral oil onto the surface of the monolayer. Each intercellular calcium wave was scored using a standardized grid, and points were assigned depending upon the distance the wave traveled and the fluorescent intensity observed within each region. Experiments were performed in the presence of inhibitors of gap junctions and connexin hemichannels (octanol), ATP (apyrase and MDL 12330 A), prostaglandins (indomethacin, high concentrations of lanthanum), the prostaglandin transporter, PGT (DIDS), and transmembrane calcium flux (low concentrations of lanthanum). Octanol, apyrase and MDL 12330 A failed to modify the far waves, indicating gap junctions, connexin hemichannels and ATP do not participate in the paracrine mechanism. Indomethacin at 30, 100 and 300 microM, in a dose dependent manner, reduced the far wave score to 0, suggesting a prostaglandin was critically involved in the mechanism. DIDS reduced the far wave score, but did not fully inhibit wave propagation, suggesting the presence of PGT-dependent and -independent components to the mechanism. Lanthanum at 0.1 mM had no effect, but at 1 mM, reduced the far wave score. These results are consistent with PGF2alpha and/or PGE2 being the signal molecule for the PGT-dependent component. Taken together, these data indicate that long distance intercellular calcium waves in cultured human myocytes utilizes a paracrine signaling mechanism, but with more than one extracellular signaling compound.