Water permeation drives tumor cell migration in confined microenvironments.

Cell migration is a critical process for diverse (patho)physiological phenomena. Intriguingly, cell migration through physically confined spaces can persist even when typical hallmarks of 2D planar migration, such as actin polymerization and myosin II-mediated contractility, are inhibited. Here, we present an integrated experimental and theoretical approach ("Osmotic Engine Model") and demonstrate that directed water permeation is a major mechanism of cell migration in confined microenvironments. Using microfluidic and imaging techniques along with mathematical modeling, we show that tumor cells confined in a narrow channel establish a polarized distribution of Na+/H+ pumps and aquaporins in the cell membrane, which creates a net inflow of water and ions at the cell leading edge and a net outflow of water and ions at the trailing edge, leading to net cell displacement. Collectively, this study presents an alternate mechanism of cell migration in confinement that depends on cell-volume regulation via water permeation.

Self-healing flexible strain sensor fabricated through 3D printing template sacrifice for motion monitoring with enhanced healing and mechanical performance.

With the advancements in flexible materials and information technology, flexible sensors are becoming increasingly pervasive in various aspects of life and production. They hold immense potential for further development in areas such as motion detection, electronic skin, soft robots, and wearable devices. Aminopropyl-terminated polydimethylsiloxane (PDMS) was used as the raw material, while a diisocyanate reagent served as the cross-linking agent for the polymerization reaction, which involved the introduction of ureido groups, containing N-H and C=O bonds, into the long siloxane chain. The dynamic hydrogen bonding between the clusters completes the self-healing of the material. Using 1-[3-(trimethoxysilyl)propyl]urea as a grafting agent, the urea groups are introduced into graphene oxide and carbon nanotubes (CNTs) as conductive fillers. Subsequently, a flexible polymer is used as the substrate to prepare conductive flexible self-healing composites. By controlling the amount of conductive fillers, flexible strain materials with varying sensitivities are obtained. Design the structure of the flexible strain sensor using three-dimensional (3D) modeling software with deposition printing method.

Spontaneous symmetry-breaking of the active cluster drives the directed movement and self-sustained oscillation of symmetric rod-like passive particles.

Active particles without detailed balance can rectify their random motions to drive the directed movement or rotation of asymmetric passive obstacles. However, whether they can drive the directed movement of symmetric passive obstacles is still unclear. Here, we show that a rod-like passive particle which is fixed to move along the x-axis in an active bath can keep long-lived directed movement at nearly constant speed due to the spontaneous symmetry breaking of the neighboring active particle cluster. If the passive particle is further confined by a harmonic potential, it may undergo self-sustained periodic oscillation for an appropriate length of the passive particle and self-propelled velocity of active particles. The restoring force from the harmonic potential will trigger the velocity jump-off and thus lead to self-sustained periodic oscillation. Remarkably, the relationship between the velocity of the passive particle and the external force shows that the effective viscosity of the active bath may become negative in some regime. Finally, we develop a minimum 1D theoretical model to further probe the mechanism underlying the directed movement and self-sustained oscillation of the passive particle. Our findings reveal the effect of the moving boundary on the active bath and demonstrate a novel method to extract practical mechanical work from the active bath to propel microdevices.

Intercellular water exchanges trigger soliton-like waves in multicellular systems.

Oscillations and waves are ubiquitous in living cellular systems. Generations of these spatiotemporal patterns are generally attributed to some mechanochemical feedbacks. Here, we treat cells as open systems, i.e., water and ions can pass through the cell membrane passively or actively, and reveal a new origin of wave generation. We show that osmotic shocks above a shock threshold will trigger self-sustained cell oscillations and result in long-range waves propagating without decrement, a phenomenon that is analogous to the excitable medium. The traveling wave propagates along the intercellular osmotic pressure gradient, and its wave speed scales with the magnitude of intercellular water flows. Furthermore, we also find that the traveling wave exhibits several hallmarks of solitary waves. Together, our findings predict a new mechanism of wave generation in living multicellular systems. The ubiquity of intercellular water exchanges implies that this mechanism may be relevant to a broad class of systems.

Electromechanical model for object roughness perception during finger sliding.

Touch allows us to gather abundant information in the world around us. However, how sensory cells embedded in the fingers convey texture information into their firing patterns is still poorly understood. Here, we develop an electromechanical model for roughness perception by incorporating main ingredients such as voltage-gated ion channels, active ion pumps, mechanosensitive channels, and cell deformation. The model reveals that sensory cells can convey texture wavelengths into the period of their firing patterns as the finger slides across object surfaces, but they can only convey a limited range of texture wavelengths. We also show that an increase in sliding speed broadens the decoding wavelength range at the cost of reduction of lower perception limits. Thus, a smaller sliding speed and a bigger contact force may be needed to successfully discern a smooth surface, consistent with previous psychophysical observations. Moreover, we show that cells with slowly adapting mechanosensitive channels can still fire action potentials under static loadings, indicating that slowly adapting mechanosensitive channels may contribute to the perception of coarse textures under static touch. Our work thus provides a new theoretical framework to study roughness perception and may have important implications for the design of electronic skin, artificial touch, and haptic interfaces.

Activation of Topological Defects Induces a Brittle-to-Ductile Transition in Epithelial Monolayers.

Epithelial monolayers are subjected to various mechanical forces, such as stretching, shearing, and compression. Thus, its mechanical response to external loadings is essential for its biological functions. However, the mechanism of the fracture failure of the epithelial monolayer remains poorly understood. Here, by introducing a new type of topological transition, i.e., detach transition or T4 transition, we develop a modified cellular vertex model to investigate the rupture of the cell monolayer. Interestingly, we find a brittle-to-ductile transition in epithelial monolayers, which is controlled by the mechanical properties of single cells and cell-cell contacts. We reveal that the external loadings can activate cell rearrangement in ductile cell monolayers. The plastic deformation results from the nucleation and propagation of "pentagon-heptagon defects" in analogy with the topological defects commonly seen in 2D materials. By using a simplified four-cell model, we further demonstrate that the brittle-to-ductile transition is induced by the competition between cell rearrangement and cell detachment. Our work provides a new theoretical framework to study the rupture of living tissues and may have important implications for many other biological processes, such as wound healing and tissue morphogenesis.

DC electric field-driven heartbeat phenomenon of gallium-based liquid metal on a floating electrode.

The heart beating phenomenon of room temperature liquid metal (LM) mercury has attracted much attention in the past years, but its research and application are limited because of the low vapor pressure and high toxicity. Here, a fundamental scientific finding is reported that the non-toxic eutectic gallium indium (EGaIn) alloy droplets beat periodically at a certain frequency based on a floating electrode under the stimulation of the direct current (DC) field. The essential characteristics of heart beating are the displacement and the projected area change of the LM droplet. The mechanism of this phenomenon is the self-regulation of interfacial tension caused by chemical oxidation, chemical corrosion, and continuous electrowetting. In this article, a series of experiments are also carried out to examine the effects of different factors on the heartbeat, such as voltage, the volume of the droplet, the droplet immersion depth, the electrolyte solution concentration, the distance of electrodes, and the type of floating electrode. Finally, the heartbeat state and application boundary of the LM droplet under different conditions are summarized by imitating the human life process. The periodic changes of the LM droplet under an external DC electric field provide a new method to simulate the beating of the heart artificially, and can be applied to the research of organ chip fluid pumping in the future.

Mechanical properties of external confinement modulate the rounding dynamics of cells.

Many studies have demonstrated that mitotic cells can round up against external impediments. However, how the stiffness of external confinement affects the dynamics of rounding force/pressure and cell volume remains largely unknown. Here, we develop a theoretical framework to study the rounding of adherent cells confined between a substrate and a cantilever. We show that the rounding force and pressure increase exclusively with the effective confinement on the cell, which is related to the cantilever stiffness and the separation between cantilever and substrate. Remarkably, an increase of cantilever stiffness from 0.001 to 1 N/m can lead to a 100-fold change in rounding force. This model also predicts an active role of confinement stiffness in regulating the dynamics of cell volume and hydrostatic pressure. We find that the dynamic changes of cellular volume and hydrostatic pressure after osmotic shocks are opposite if the cantilever is soft, whereas the dynamic changes of cellular volume and pressure are the same if the cantilever is stiff. Taken together, this work demonstrates that confinement stiffness appears as a critical regulator in regulating the dynamics of rounding force and pressure. Our findings also indicate that the difference in cantilever stiffness need to be considered when comparing the measured rounding force and pressure from various experiments.

Endocytosis and exocytosis protect cells against severe membrane tension variations.

The ability of cells to regulate their shape and volume is critical for many cell functions. How endocytosis and exocytosis, as important ways of membrane trafficking, affect cellular volume regulation is still unclear. Here, we develop a theoretical framework to study the dynamics of cell volume, endocytosis, and exocytosis in response to osmotic shocks and mechanical loadings. This model can not only explain observed dynamics of endocytosis and exocytosis during osmotic shocks but also predict the dynamics of endocytosis and exocytosis during cell compressions. We find that a hypotonic shock stimulates exocytosis, while a hypertonic shock stimulates endocytosis; and exocytosis in turn allows cells to have a dramatic change in cell volume but a small change in membrane tension during hyposmotic swelling, protecting cells from rupture under high tension. In addition, we find that cell compressions with various loading speeds induce three distinct dynamic modes of endocytosis and exocytosis. Finally, we show that increasing endocytosis and exocytosis rates reduce the changes in cell volume and membrane tension under fast cell compression, whereas they enhance the changes in cell volume and membrane tension under slow cell compression. Together, our findings reveal critical roles of endocytosis and exocytosis in regulating cell volume and membrane tension.

Characterization of Particle Movement and High-Resolution Separation of Microalgal Cells via Induced-Charge Electroosmotic Advective Spiral Flow.

Microalgae are renewable, sustainable, and economical sources of biofuels and are capable of addressing pressing global demand for energy security. However, two challenging issues to produce high-level biofuels are to separate promising algal strains and protect biofuels from contamination of undesired bacteria, which rely on an economical and high-resolution separation technology. Separation technology based on induced-charge electroosmotic (ICEO) vortices offers excellent promise in economical microalga separation for producing biofuels because of its reconfigurable and flexible profiles and sensitive and precise selectivity. In this work, a practical ICEO vortex device is developed to facilitate high-resolution isolation of rich-lipid microalgae for the first time. We investigate electrokinetic equilibrium states of particles and particle-fluid ICEO effect in binary-particle manipulation. Nanoparticle separation is performed to demonstrate the feasibility and resolution of this device, yielding clear separation. Afterward, we leverage this technology in isolation of Chlorella vulgaris from heterogeneous microalgae with the purity exceeding 96.4%. Besides, this platform is successfully engineered for the extraction of single-cell Oocystis sp., obtaining the purity surpassing 95.2%. Moreover, with modulating parameters, we isolate desired-cell-number Oocystis sp. enabling us to investigate proliferation mode and carry out transcriptome analyses of Oocystis sp. for high-quality neutral lipids. This platform can be extended directly to economically separate other biological micro/nanosamples to address pressing issues, involving energy security, environmental monitoring, and disease diagnosis.

Flexible Microswimmer Manipulation in Multiple Microfluidic Systems Utilizing Thermal Buoyancy-Capillary Convection.

Flexible and accurate control of microswimmers is significant for lots of applications. Herein, we present a method for effective microswimmer manipulation in multiple microfluidic systems by thermal buoyancy-capillary convection. In the microdevice, four strips of microheaters arranged at the bottom of the microchannel are used to unevenly heat microfluids, and the convection flow forms under the influence of gravity and interfacial tension gradient. By adjusting the DC signals applied on these four heating elements, the intensity and direction of convection flow can be flexibly adjusted. Accordingly, granular samples dispersed in liquid buffer can be controllably driven to the target position by the Stokes drag. The swimming behavior of polystyrene (PS) microspheres at the solid-liquid interface of the device is first investigated. It shows that the PS microswimmers can migrate along various geometrical patterns by powering the microheaters with designed voltage combinations, and the migration velocity is positively affected by the increased voltage. Then, the butyl acrylate (BA) microswimmers are manipulated at the gas-liquid interface of the microchip. It turns out that the BA microswimmers migrate oppositely compared with PS swimmers under the same energization strategy. Additionally, the translation direction of BA swimmers can be changed over a 360° range by different voltage combinations. The multifunctionality of our approach is further demonstrated by conveniently driving the trimethylolpropane triacrylate microswimmers at the liquid-liquid interface of the microplatform along different directions and pathlines. Therefore, this technique can be promising for many cases needing granular sample control, such as cargo delivery and sensing.

Dielectric Characterization and Multistage Separation of Various Cells via Dielectrophoresis in a Bipolar Electrode Arrayed Device.

Isolation of microalgal cells is as an indispensable part of producing biofuels for energy security and detecting toxic contaminants for marine routine monitoring. Microalgae live together with various microalgae naturally, and abundant samples need to be tackled in practical applications. Therefore, effective separation technologies need to be developed urgently to achieve high-throughput separation of various microalgae. Herein, we develop a reliable device to characterize the dielectric response of microalgae and sequentially separate various microalgae utilizing dielectrophoretic force in a bipolar electrode (BPE) arrayed device. First, by investigating the array width extension (AWE) effect on the electric- and flow-field distributions, we explore consequences of incidental electrohydrodynamic mechanisms and axial flow rate on the separation. Second, based on device performance on sample characterizations, we demonstrate this technology by separating microparticles in three- and five-channel devices. Third, we discriminate dead and live cells to explore its capability using the cell viability test and illustrate the AWE influence on the separation. Fourth, we characterize dielectric responses of different microalgae and separate C. vulgaris and Oocystis sp. Finally, we extended BPEs in length and developed an arrayed device for sequential separation of various microalgae, and this platform is successfully engineered in high-throughput isolation of C. vulgaris from complex samples. This technology presents good potential in addressing depleting fossil fuel and burgeoning environmental concerns due to its performance in the separation of microalgal strains from complex samples.

Label-Free Multitarget Separation of Particles and Cells under Flow Using Acoustic, Electrophoretic, and Hydrodynamic Forces.

Multiplex separation of mixed biological samples is essential in a considerable portion of biomedical research and clinical applications. An automated and operator-independent process for the separation of samples is highly sought after. There is a significant unmet need for methods that can perform fractionation of small volumes of multicomponent mixtures. Herein, we design an integrated chip that combines acoustic and electric fields to enable efficient and label-free separation of multiple different cells and particles under flow. To facilitate the connection of multiple sorting mechanisms in tandem, we investigate the electroosmosis (EO)-induced deterministic lateral displacement (DLD) separation in a combined pressure- and DC field-driven flow and exploit the combination of the bipolar electrode (BPE) focusing and surface acoustic wave (SAW) sorting modules. We successfully integrate four sequential microfluidic modules for multitarget separation within a single platform: (i) sorting particles and cells relying on the size and surface charge by adjusting the flow rate and electric field using a DLD array; (ii) alignment of cells or particles within a microfluidic channel by a bipolar electrode; (iii) separation of particles based on compressibility and density by the acoustic force; and (iv) separation of viable and nonviable cells using dielectric properties via the dielectrophoresis (DEP) force. As a proof of principle, we demonstrate the sorting of multiple cell and particle types (polystyrene (PS) particles, oil droplets, and viable and nonviable yeast cells) with high efficiency. This integrated microfluidic platform combines multiple functional components and, with its ability to noninvasively sort multiple targeted cells in a label-free manner relying on different properties, is compatible with high-definition imaging, showing great potential in diverse diagnostic and analysis applications.

Bone Measures by Dual-Energy X-Ray Absorptiometry and Peripheral Quantitative Computed Tomography in Young Women With Type 1 Diabetes Mellitus.

Understanding bone fragility in young adult females with type 1 diabetes mellitus (T1DM) is of great clinical importance since the high fracture risk in this population remains unexplained. This study aimed to investigate bone health in young adult T1DM females by comparing relevant variables determined by dual-energy X-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT) at the tibia and pQCT-based finite element analysis (pQCT-FEA) between T1DM subjects (n = 21) and age-, height- and weight-matched controls (n = 63). Tibial trabecular density (lower by 7.1%; 228.8 ± 33.6 vs 246.4 ± 31.8 mg/cm3, p = 0.02) and cortical thickness (lower by 7.3%; 3.8 ± 0.5 vs 4.1 ± 0.5 cm, p = 0.03) by pQCT were significantly lower in T1DM subjects than in controls. Tibial shear stiffness by pQCT-FEA was also lower in T1DM subjects than in controls at both the 4% site (by 17.1%; 337.4 ± 75.5 vs 407.1 ± 75.4 kN/mm, p < 0.01) and 66% site (by 7.9%; 1113.0 ± 158.6 vs 1208.8 ± 161.8 kN/mm, p = 0.03). These differences remained statistically significant after adjustment for confounding factors. No difference between groups was observed in DXA-determined variables (all p ≥ 0.08), although there was a trend towards lower aBMD at the lumbar spine in T1DM subjects than in controls after adjustment for confounders (p = 0.053). These novel findings elicited using pQCT and pQCT-FEA suggest a clinically significant impact of T1DM on bone strength in young adult females with T1DM. Peripheral QCT and pQCT-FEA may provide more information than DXA alone on bone fragility in this population. Further longitudinal studies with a larger sample size are warranted to understand the evolution and causes of bone fragility in young T1DM females.

A Refined Composite Multivariate Multiscale Fluctuation Dispersion Entropy and Its Application to Multivariate Signal of Rotating Machinery.

In the fault monitoring of rotating machinery, the vibration signal of the bearing and gear in a complex operating environment has poor stationarity and high noise. How to accurately and efficiently identify various fault categories is a major challenge in rotary fault diagnosis. Most of the existing methods only analyze the single channel vibration signal and do not comprehensively consider the multi-channel vibration signal. Therefore, this paper presents Refined Composite Multivariate Multiscale Fluctuation Dispersion Entropy (RCMMFDE), a method which extracts the recognition information of multi-channel signals with different scale factors, and the refined composite analysis ensures the recognition stability. The simulation results show that this method has the characteristics of low sensitivity to signal length and strong anti-noise ability. At the same time, combined with Joint Mutual Information Maximisation (JMIM) and support vector machine (SVM), RCMMFDE-JMIM-SVM fault diagnosis method has been proposed. This method uses RCMMFDE to extract the state characteristics of the multiple vibration signals of the rotary machine, and then uses the JMIM method to extract the sensitive characteristics. Finally, different states of the rotary machine are classified by SVM. The validity of the method is verified by the composite gear fault data set and bearing fault data set. The diagnostic accuracy of the method is 99.25% and 100.00%. The experimental results show that RCMMFDE-JMIM-SVM can effectively recognize multiple signals.

Durotaxis Index of 3T3 Fibroblast Cells Scales with Stiff-to-Soft Membrane Tension Polarity.

Cell durotaxis is an essential process in tissue development. Although the role of cytoskeleton in cell durotaxis has been widely studied, whether cell volume and membrane tension are implicated in cell durotaxis remains unclear. By quantifying the volume distribution during cell durotaxis, we show that the volume of 3T3 fibroblast cells decreases by almost 40% as cells migrate toward stiffer regions of gradient gels. Inhibiting ion transporters that can reduce the amplitude of cell volume decrease significantly suppresses cell durotaxis. However, from the correlation analysis, we find that durotaxis index does not correlate with the cell volume decrease. It scales with the membrane tension difference in the direction of stiffness gradient. Because of the tight coupling between cell volume and membrane tension, inhibition of Na+/K+ ATPase and Na+/H+ exchanger results in smaller volume decrease and membrane tension difference. Collectively, our findings indicate that the polarization of membrane tension is a central regulator of cell durotaxis, and Na+/K+ ATPase and Na+/H+ exchanger can help to maintain the membrane tension polarity.

Flexible Particle Focusing and Switching in Continuous Flow via Controllable Thermal Buoyancy Convection.

We present a novel approach that utilizes thermal buoyancy convection to achieve flexible particle focusing and switching in continuous flow of a microfluidic system. In this platform, three strip microheaters, A, B, and C, are symmetrically distributed at the bottom of microchannel, and they are isolated from the particle suspension by a thin glass slide. Continual transverse convection flow forms when the microheaters are energized by dc signals. The flow patterns are readily tuned by changing the energization strategies of the microheater array, leading to the modulation of the position of flow stagnation region. Accordingly, microparticles dispersed in fluids are rapidly focused to the flow stagnation region by the Stokes drag and thus form a continuous particle beam. The particle beam can also be switched to different lateral positions by adjusting the control voltages. This particle manipulation method is first demonstrated by respectively energizing these three microheaters and subsequently switching silica particles into different outlets. The lateral position of the particle beam then is flexibly controlled by simultaneously energizing microheaters A and B (or B and C) and adjusting the voltage applied on microheater A (or C). Furthermore, the versatility of this approach is proved by focusing and switching of microsized droplets, that is, oil-in-water and water-in-oil-in-water emulsion droplets. Finally, we use poly(ethylene glycol) diacrylate microgels, excellent reactant carriers, as an experimental sample and flexibly manipulate them in this microdevice, demonstrating this strategy's applicability for the cargo delivery. Therefore, this technique can be attractive for many particle preprocessing applications.

Different-shaped micro-objects driven by active particle aggregations.

The dynamics of passive micro-objects in an active bath has been receiving much attention. However, the influence of the shapes of micro-objects remains unclear. Here, we use 2D simulation to investigate the interaction between active Brownian particles and different-shaped passive micro-objects. We show that active particles accumulate around micro-objects and self-assemble into living aggregations at a high active velocity and high volume fraction. The shapes of micro-objects affect the distributions of the aggregations. In turn, the different distribution of aggregations influences the motion of micro-objects and induces abnormal diffusive behaviors. We further demonstrate that polar distributed aggregations at a high active velocity and the inhibition of the active bath at a low active velocity induce the counterintuitive anisotropic enhanced diffusion of rods, and the steric interaction between active particles induces the reverse translation-rotation coupled diffusion of chevrons.

The inhibition of concentrated active baths.

Passive tracers in the active bath express fascinating behaviors. However, most studies are restricted to dilute active baths. Here, we use 2D simulation of suspensions consisting of active Brownian particles and a passive disk-shaped tracer to investigate tracers' diffusive behaviors in a wide range of volume fractions. Due to the competition between the thermal noise and collisions with active particles, tracers express a first transition from the normal diffusion to the superdiffusion at a short time scale and recur to normal diffusion at a long time scale. At a low volume fraction, infrequent active collisions retard the first transition of smaller tracers. At a high volume fraction, active particles with high activity aggregating around tracers induce a bimodal probability distribution function of tracer displacements during superdiffusion. Considering the enhancement of diffusion, the non-dimensional enhanced diffusivity increases asymptotically with the Peclet number. The asymptotic line gives an upper limit of non-dimensional enhanced diffusivity of tracers. Cases with lower enhanced diffusion have a high volume fraction and a low active velocity that indicates the inhibition of concentrated active baths. With the high negentropic work of these cases, the inhibition is explained as the change of the configuration of active baths for introducing tracers.

Number of Removed Pelvic Lymph Nodes as a Prognostic Marker in FIGO Stage IB1 Cervical Cancer with Negative Lymph Nodes.

STUDY OBJECTIVE: To investigate whether the number of removed lymph nodes (RLNs) influences the clinical outcome of stage IB1 cervical cancer on the premise of uniform pelvic lymphadenectomy. DESIGN: Retrospective cohort study. SETTING: Obstetrics and Gynecology Hospital of Fudan University. PATIENTS: Women (n = 782) with stage IB1 cervical cancer. INTERVENTIONS: Laparoscopic radical hysterectomy and uniform pelvic lymphadenectomy (common iliac, external iliac, internal iliac, obturator) for stage IB1 cervical cancer. The median time of follow-up was 64.7 months (range, 4.3-102.8). MEASUREMENTS AND MAIN RESULTS: Of 782 patients with stage IB1 cervical cancer, the median number of pelvic RLNs was 19 (range, 7-49). Twenty-one patients (2.7%) had RLNs ≤ 10, 461 (59.0%) had 11 to 20, 263 (33.6%) had 21 to 30, and 37 (4.7%) had RLNs ≥ 31. The differences were not statistically significant in the clinicopathologic characteristics between the 4 groups (p >.05). In the multivariate analysis, pelvic RLN number became an independent prognostic factor for progression-free survival (PFS) and cancer-specific survival (CSS) in stage IB1 cervical cancer (p = .029; .015 for CSS and PFS). After the stratified analysis by lymph node metastasis, RLN number remained an independent predictive value (p = .026 for CSS, p = .012 for PFS) in patients with negative pelvic lymph nodes. Moreover, patients with RLN number ≤ 10 carried a 5.550-fold higher risk for progression (p <.001) and 5.596-fold greater likelihood of death (p = .001) than those with RLN number > 10 in case of no lymph node metastasis. CONCLUSION: With uniform pelvic lymphadenectomy, the total pelvic RLN number could be a valuable predictor of outcome in stage IB1 cervical cancer without lymph node metastasis during a follow-up of at least 5 years.