Monitoring uterine contractions during labor: current challenges and future directions.

Organ-level models are used to describe how cellular and tissue-level contractions coalesce into clinically observable uterine contractions. More importantly, these models provide a framework for evaluating the many different contraction patterns observed in laboring patients, ideally offering insight into the pitfalls of currently available recording modalities and suggesting new directions for improving recording and interpretation of uterine contractions. Early models proposed wave-like propagation of bioelectrical activity as the sole mechanism for recruiting the myometrium to participate in the contraction and increase contraction strength. However, as these models were tested, the results consistently revealed that sequentially propagating waves do not travel long distances and do not encompass the gravid uterus. To resolve this discrepancy, a model using 2 mechanisms, or a "dual model," for organ-level signaling has been proposed. In the dual model, the myometrium is recruited by action potentials that propagate wave-like as far as 10 cm. At longer distances, the myometrium is recruited by a mechanotransduction mechanism that is triggered by rising intrauterine pressure. In this review, we present the influential models of uterine function, highlighting their main features and inconsistencies, and detail the role of intrauterine pressure in signaling and cervical dilation. Clinical correlations demonstrate the application of organ-level models. The potential to improve the recording and clinical interpretation of uterine contractions when evaluating labor is discussed, with emphasis on uterine electromyography. Finally, 7 questions are posed to help guide future investigations on organ-level signaling mechanisms.

Mid-trimester uterine electromyography in patients with a short cervix.

BACKGROUND: Preterm birth is the largest single cause of infant death in the United States. A cervical length of <2.5 cm, measured in the mid-trimester, has been shown to identify individuals at increased risk. Uterine electromyography is an emerging technology for noninvasively assessing uterine bioelectrical activity. With its ability to characterize nuanced differences in myometrial signals, uterine electromyography assessments during the mid-trimester may provide insight into the mechanisms of cervical shortening. OBJECTIVE: This study aimed to characterize uterine bioelectrical activity in pregnant individuals with short cervices in the mid-trimester compared with that of pregnant individuals of the same gestational age with normal cervical lengths. STUDY DESIGN: This is a prospective cohort study of subjects with singleton, nonanomalous pregnancies between 16 weeks and 0 days and 22 weeks and 6 days of gestational age. Subjects with normal cervical length (≥3.0 cm) were compared with subjects with short cervical length (<2.5 cm). The short-cervical-length cohort was further stratified by history of preterm birth. Multichannel uterine electromyography recordings were obtained for ∼60 minutes using proprietary, directional electromyography sensors on the abdomen. Uterine electromyography signals were observed and classified in groups as spikes, short bursts, and bursts. Primary outcomes were relative expression of spike, short-burst, and burst uterine electromyography signals. Subgroup analyses assessed each signal percentage by cervical length, history of preterm birth, and gestational age at delivery. Differences in percentage of uterine electromyography signals according to cervical length were analyzed using nonparametric tests of significance. RESULTS: Of the 28 included subjects, 10 had normal and 18 had short cervical length. There were 9 subjects with short cervical length and a history of preterm birth. Spikes were the most commonly recorded signals and were higher in the normal-cervical-length cohort (96.3% [interquartile range, 93.1%-100.0%]) than the short-cervical-length cohort (75.2% [interquartile range, 66.7%-92.0%], P=.001). In contrast, median percentages of short-bursts and bursts were significantly higher in subjects with a short cervical length (17.3% [interquartile range, 13.6%-23.9%] vs 2.5% for normal cervical length [interquartile range, 0%-5.5%], P=.001 and 6.6% [interquartile range, 0%-13.4%] vs 0% for normal cervical length [interquartile range, 0%-2.8%], P=.014, respectively). Within subgroup analyses, cervical length was inversely proportional to percentage of observed short-bursts (P=.013) and bursts (P=.014). Subjects with short cervical length and history of preterm birth had higher burst percentages (12.8% [interquartile range, 9.0%-15.7%]) than those with short cervical length and no history of preterm birth (3.3% [interquartile range, 0%-5.0%], P=.003). CONCLUSION: Short-burst and burst uterine electromyography signals are observed more frequently in mid-trimester patients with short cervical lengths. This relationship provides insight into abnormal myometrial activation in the mid-trimester and offers a plausible biophysiological link to cervical shortening.

Mechanotransduction mechanisms for coordinating uterine contractions in human labor.

This review presents evolving concepts of how the human uterus contracts in pregnancy, with emphasis on the mechanisms of long-distance signaling. Action potential propagation has historically been assumed to be the sole mechanism for signaling and tissue recruitment over both short and long distances. However, data in animals and humans indicate that a single action potential does not travel distances greater than a few centimeters. To address this enigma, a long-distance signaling mechanism based on hydraulic signaling and mechanotransduction is developed. By combining this mechanism for long-distance signaling with the action potential propagation mechanism for signaling over short distances, a comprehensive dual mechanism model (or 'dual model') of uterine function is formulated. Mechanotransduction is an accepted phenomenon of myometrium, but the dual model identifies mechanotransduction as relevant to normal labor. For hydraulic signaling, a local contraction slightly increases intrauterine pressure, which globally increases wall tension. Increased wall tension then mechanically induces additional local contractions that further raise pressure. This leads to robust, positive feedback recruitment that explains the emergence of consistently strong contractions of human labor. Three key components of the dual model - rapid long-distance signaling, mechanical triggering, and electrical activity - converge with the concept of mechanically sensitive electrogenic pacemakers distributed throughout the wall. The dual model retains excitation-contraction coupling and action potential propagation for signaling over short distances (<10cm) and hence is an extension of the action potential model rather than a replacement.

Why the heart is like an orchestra and the uterus is like a soccer crowd.

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.

Uterine overdistention induces preterm labor mediated by inflammation: observations in pregnant women and nonhuman primates.

OBJECTIVE: Uterine overdistention is thought to induce preterm labor in women with twin and multiple pregnancies, but the pathophysiology remains unclear. We investigated for the first time the pathogenesis of preterm birth associated with rapid uterine distention in a pregnant nonhuman primate model. STUDY DESIGN: A nonhuman primate model of uterine overdistention was created using preterm chronically catheterized pregnant pigtail macaques (Macaca nemestrina) by inflation of intraamniotic balloons (N = 6), which were compared to saline controls (N = 5). Cesarean delivery was performed due to preterm labor or at experimental end. Microarray, quantitative reverse transcriptase polymerase chain reaction, Luminex (Austin, TX), and enzyme-linked immunosorbent assay were used to measure messenger RNA (mRNA) and/or protein levels from monkey (amniotic fluid, myometrium, maternal plasma) and human (amniocytes, amnion, myometrium) tissues. Statistical analysis employed analysis of covariance and Wilcoxon rank sum. Biomechanical forces were calculated using the law of Laplace. RESULTS: Preterm labor occurred in 3 of 6 animals after balloon inflation and correlated with greater balloon volume and uterine wall stress. Significant elevations of inflammatory cytokines and prostaglandins occurred following uterine overdistention in an "inflammatory pulse" that correlated with preterm labor (interleukin [IL]-1ß, tumor necrosis factor [TNF]-α, IL-6, IL-8, CCL2, prostaglandin E2, prostaglandin F2α, all P < .05). A similar inflammatory response was observed in amniocytes in vitro following mechanical stretch (IL1ß, IL6, and IL8 mRNA multiple time points, P < .05), in amnion of women with polyhydramnios (IL6 and TNF mRNA, P < .05) and in amnion (TNF-α) and myometrium of women with twins in early labor (IL6, IL8, CCL2, all P < .05). Genes differentially expressed in the nonhuman primate after balloon inflation and in women with polyhydramnios and twins are involved in tissue remodeling and muscle growth. CONCLUSION: Uterine overdistention by inflation of an intraamniotic balloon is associated with an inflammatory pulse that precedes and correlates with preterm labor. Our results indicate that inflammation is an early event after a mechanical stress on the uterus and leads to preterm labor when the stress is sufficiently great. Further, we find evidence of uterine tissue remodeling and muscle growth as a common, perhaps compensatory, response to uterine distension.

Linking myometrial physiology to intrauterine pressure; how tissue-level contractions create uterine contractions of labor.

The mechanisms used to coordinate uterine contractions are not known. We develop a new model based on the proposal that there is a maximum distance to which action potentials can propagate in the uterine wall. This establishes "regions", where one action potential burst can rapidly recruit all the tissue. Regions are recruited into an organ-level contraction via a stretch-initiated contraction mechanism (myometrial myogenic response). Each uterine contraction begins with a regional contraction, which slightly increases intrauterine pressure. Higher pressure raises tension throughout the uterine wall, which initiates contractions of more regions and further increases pressure. The positive feedback synchronizes regional contractions into an organ-level contraction. Cellular automaton (CA) simulations are performed with Mathematica. Each "cell" is a region that is assigned an action potential threshold. An anatomy sensitivity factor converts intrauterine pressure to regional tension through the Law of Laplace. A regional contraction occurs when regional tension exceeds regional threshold. Other input variables are: starting and minimum pressure, burst and refractory period durations, enhanced contractile activity during an electrical burst, and reduced activity during the refractory period. Complex patterns of pressure development are seen that mimic the contraction patterns observed in laboring women. Emergent behavior is observed, including global synchronization, multiple pace making regions, and system memory of prior conditions. The complex effects of nifedipine and oxytocin exposure are simulated. The force produced can vary as a nonlinear function of the number of regions. The simulation directly links tissue-level physiology to human labor. The concept of a uterine pacemaker is re-evaluated because pace making activity may occur well before expression of a contraction. We propose a new classification system for biological CAs that parallels the 4-class system of Wolfram. However, instead of classifying the rules, biological CAs should classify the set of input values for the rules that describe the relevant biology.

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.

Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition.

An incomplete understanding of the molecular events that regulate the myometrial transition from the quiescent pregnant state to the active contractile state during labor has hindered the development of improved therapies for preterm labor. During myometrial activation, proteins that prime the smooth muscle for contraction are upregulated, allowing maximal responsiveness to contractile agonists and thereby producing strong phasic contractions. Upregulation of one such protein, COX-2, generates PGs that induce contractions. Intriguingly, the predominant myometrial PG produced just prior to labor is prostacyclin (PGI2), a smooth muscle relaxant. However, here we have shown that activation of PGI2 receptor (IP) upregulated the expression of several contractile proteins and the gap junction protein connexin 43 through cAMP/PKA signaling in human myometrial tissue in organ and cell culture. Functionally, these IP-dependent changes in gene expression promoted an enhanced contractile response to oxytocin in pregnant human myometrial tissue strips, which was inhibited by the IP antagonist RO3244794. Furthermore, contractile protein induction was dependent on the concentration and time of exposure to the PGI2 analog iloprost and was blocked by both RO3244794 and PKA knockdown. We therefore propose that PGI2-mediated upregulation of contractile proteins and connexin 43 is a critical step in myometrial activation, allowing for a maximal contractile response. Our observations have important implications regarding activation of the myometrium prior to the onset of labor.

Evaluating aminophylline and progesterone combination treatment to modulate contractility and labor-related proteins in pregnant human myometrial tissues.

Progesterone (P4) and cyclic adenosine monophosphate (cAMP) are regarded as pro-quiescent factors that suppress uterine contractions during pregnancy. We previously used human primary cells in vitro and mice in vivo to demonstrate that simultaneously enhancing myometrial P4 and cAMP levels may reduce inflammation-associated preterm labor. Here, we assessed whether aminophylline (Ami; phosphodiesterase inhibitor) and P4 can reduce myometrial contractility and contraction-associated proteins (CAPs) better together than individually; both agents are clinically used drugs. Myometrial tissues from pregnant non-laboring women were treated ex vivo with Ami acutely (while spontaneous contracting) or throughout 24-h tissue culture (±P4); isometric tension measurements, PKA assays, and Western blotting were used to assess tissue contractility, cAMP action, and inflammation. Acute (1 h) treatment with 250 and 750 µM Ami reduced contractions by 50% and 84%, respectively, which was not associated with a directly proportional increase in whole tissue PKA activity. Sustained myometrial relaxation was observed during 24-h tissue culture with 750 µM Ami, which did not require P4 nor reduce CAPs. COX-2 protein can be reduced by 300 nM P4 but this did not equate to myometrial relaxation. Ami (250 µM) and P4 (100 and 300 nM) co-treatment did not prevent oxytocin-augmented contractions nor reduce CAPs during interleukin-1ß stimulation. Overall, Ami and P4 co-treatment did not suppress myometrial contractions more than either agent alone, which may be attributed to low specificity and efficacy of Ami; cAMP and P4 action at in utero neighboring reproductive tissues during pregnancy should also be considered.

Myocytes, myometrium, and uterine contractions.

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.

High prevalence of thyroid disease in patients with lichen sclerosus.

OBJECTIVE: To investigate the association between lichen sclerosus and thyroid disease in our patient population. STUDY DESIGN: This was a retrospective chart review of patients seen between January 1995 and September 2005 with biopsy-proven lichen sclerosus. Charts were reviewed to assess the patients' history of thyroid disease. RESULTS: We identified 211 patients with biopsy-proven lichen sclerosus, 63 (29.9%) of whom had thyroid disease. In women <55 years old, 25 of 74 (33.8%) had thyroid disease; in women > or = 55 years old, 38 of 137 (27.7%) had thyroid disease. CONCLUSION: The prevalence of thyroid disease in our patients with biopsy-proven lichen sclerosus is almost 30% and is not dependent upon age. This prevalence is 5- to 30-fold greater than in the general population.

3D Cell Culturing and Possibilities for Myometrial Tissue Engineering.

Research insights into uterine function and the mechanisms of labour have been hindered by the lack of suitable animal and cellular models. The use of traditional culturing methods limits the exploration of complex uterine functions, such as cell interactions, connectivity and contractile behaviour, as it fails to mimic the three-dimensional (3D) nature of uterine cell interactions in vivo. Animal models are an option, however, use of these models is constrained by ethical considerations as well as translational limitations to humans. Evidence indicates that these limitations can be overcome by using 3D culture systems, or 3D Bioprinters, to model the in vivo cytological architecture of the tissue in an in vitro environment. 3D cultured or 3D printed cells can be used to form an artificial tissue. This artificial tissue can not only be used as an appropriate model in which to study cellular function and organisation, but could also be used for regenerative medicine purposes including organ or tissue transplantation, organ donation and obstetric care. The current review describes recent developments in cell culture that can facilitate the development of myometrial 3D structures and tissue engineering applications.

Synchronization of regional contractions of human labor; direct effects of region size and tissue excitability.

The mechanisms used to coordinate organ-level contractions of human labor are not universally accepted. We previously proposed a dual mechanism, where electrical activity coordinates cellular contractions into tissue-level regional contractions, and mechanotransduction synchronizes the regional contractions into organ-level contractions. The simulation of this model successfully recapitulates the phasic pressure rises typical of human labor. In this work we extend the simulation to probe the effects of three critical parameters: electrical coupling (which defines functional regions within the uterine wall), enhancement of contractile responses during action potential bursts (action potential multiplier), and the threshold for mechanical recruitment of regional myometrial contractions (threshold). We test how changing the values of these parameters modulates the ability of the uterus to generate synchronized organ-level contractions. Simulations are performed using Mathematica and a non-classical cellular automaton program we recently published. At least 15 regions are necessary to generate physiologically relevant, synchronized contractions. Organ-level synchronization was improved using higher values for the action potential multiplier. At lower values of the action potential multiplier, synchronized contractions were inhibited when the number of regions was between 32 and 44, suggesting a critical level of electrical coupling is necessary at the onset of labor. Large numbers of low threshold regions resulted in contraction patterns suggestive of hyperstimulation. This work furthers support for the electrical-mechanotransduction mechanism for organ-level synchronization of uterine contractions. The mathematical simulation provides insight regarding how cellular- and tissue-level physiology converge to express synchronized contractions of human labor.

Functional separation of deep cytoplasmic calcium from subplasmalemmal space calcium in cultured human uterine smooth muscle cells.

For smooth muscle, two important functions of free intracellular calcium (Ca(2+)(i)) are modulation of plasma membrane excitability properties and modulation of the contractile apparatus. As proposed by van Breemen, Ca(2+)(i) can be divided into the subplasmalemmal space (Ca(2+)(sps)) and the deep cytosol (Ca(2+)(d)) by the superficial calcium buffer barrier. Using these distinctions, Ca(2+)(sps) activates the large conductance calcium-activated potassium channel (BK), and Ca(2+)(d) binds calcium-dependent fluorescent probes in the cytoplasm. We present here combined fluorescence-patch clamp experiments designed to simultaneously assess Ca(2+)(d) and Ca(2+)(sps) in cultured human uterine smooth muscle cells. Open probabilities (P(o)) of the BK channel were measured using the cell-attached patch clamp technique. P(o) was used to approximate changes of [Ca(2+)(sps)]. Relative concentrations of Ca(2+)(d) were approximated by observing fluorescence of Calcium green-1 (F). Under control conditions, we found similar time courses for rises of P(o) and F following 10nM oxytocin (OT) addition. In parallel experiments, but with lanthanum (La(3+)) added to the bath to block transmembrane calcium flux, P(o) was only slightly affected, but F increases were delayed and blunted. These data paradoxically indicate that following OT stimulation, the primary source of calcium for Ca(2+)(sps) is internal stores, and calcium entry from the extracellular space is required to raise Ca(2+)(d). When cells were exposed to cyclopiazonic acid (CPA) to release SR calcium stores, P(o) increased slowly, then persisted at large values. The persistence of P(o) rises suggests that removal of calcium from the subplasmalemmal space is primarily via reuptake into the SR. In the presence of La(3+), OT-induced rises of F were slightly prolonged, suggesting that transmembrane calcium flux contributes to decreasing Ca(2+)(d), but is not the primary mechanism. In summary, these data demonstrate that Ca(2+)(d) and Ca(2+)(sps) are not always intimately linked, but indicate a functional separation of the deep cytosol and the subplasmalemmal space that is consistent with the existence of a barrier to calcium diffusion between these two regions.

Sarcoplasmic reticulum, calcium waves and myometrial signalling.

Ca2+ waves are rises of intracellular free Ca2+ that occur in a temporally and spatially coordinated manner, such that a leading edge of a wave front can be clearly discerned. Waves that occur within the bounds of a single cell are termed intracellular Ca2+ waves. Generation of a Ca2+ wave may be the end result of multiple cellular signalling events and, consequently, is a mechanism of signal integration or information processing. Passage of a Ca2+ wave is a mechanism for signalling within a cell, or across a cell. This paper reviews intracellular Ca2+ waves and their relationship with the sarcoplasmic reticulum (SR) in uterine smooth muscle. Wave speeds are the rate at which the leading front of the Ca2+ wave travels, and are measured by time-lapse imaging of Ca2+-dependent fluorescent dyes. Waves can be experimentally generated in cultured cells by agonist or mechanical stimulation. Wave speeds are unaffected by the removal of Ca2+ in the bathing solution, indicating that the source of the Ca2+ is the SR. The mechanisms of intracellular wave propagation can be investigated by modulating the Ca2+ release mechanisms of the SR. The mechanism most consistent with our observations is that propagation of intracellular Ca2+ waves in cultured human uterine myocytes is SR Ca2+ release that can utilize InsP3 receptors alone, Ca2+-induced Ca2+ release receptors alone, or both together. The rate-determining step for Ca2+ wave propagation is diffusion of Ca2+ through a highly buffered cytoplasm. Passage of a Ca2+ wave also results in capacitative Ca2+ entry, which links deep cytoplasmic Ca2+ changes to subplasmalemmal Ca2+ concentrations.

Three-dimensional culture of human uterine smooth muscle myocytes on a resorbable scaffolding.

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.

Tissue-level bioelectrical signals as the trigger for uterine contractions in human pregnancy.

OBJECTIVE: To investigate the relationship between tissue-level bioelectrical signals and tension development during oxytocin-stimulated contractions of human myometrium. METHODS: We performed in vitro muscle bath experiments on human myometrial tissue strips while simultaneously monitoring bioelectrical activity with two loose-contact electrodes. Tissue was obtained by myometrial biopsy from term pregnant women at the time of cesarean delivery. Tissue strips (1 x 1 x 10 mm) were hung vertically and maintained in culture in media while suspending a 400-mg weight. The tissue exhibited strong isometric contractions in response to 5-nM oxytocin even after 10 to 14 days in culture. The electrodes were separated by 4 mm, and allowed us to distinguish between local and tissue-level bioelectrical signals. Electrical activity was monitored using two, independent AC-coupled amplifiers. RESULTS: Following exposure to oxytocin, the tissue contracted periodically every 3.5 to 6 minutes, with each contraction lasting 50 to 60 seconds. Near the beginning of each contraction, synchronized spike-like bioelectrical signals were observed in both channels. These bioelectrical signals from each electrode lasted approximately 2 seconds and demonstrated unique fingerprints that were repetitive and remarkably similar over 18 contractions. In each of the contractions, the onset of rapid force increases was synchronized with the bioelectrical signals. Cell recruitment continued during the plateau phase of each contraction even though other tissue-level bioelectrical signals were not observed. CONCLUSION: These findings suggest that the trigger for the initiation of each contraction is a tissue-level bioelectrical event, and some cells are initially recruited to participate in each contraction by excitation-contraction coupling. After the initial phase of the contraction, cells are recruited by a nonelectrical mechanism.