To understand how excitable tissues give rise to arrhythmias, it is crucially necessary to understand the electrical dynamics of cells in the context of their environment. Multicellular monolayer cultures have proven useful for investigating arrhythmias and other conduction anomalies, and because of their relatively simple structure, these constructs lend themselves to paired computational studies that often help elucidate mechanisms of the observed behavior. However, tissue cultures of cardiomyocyte monolayers currently require the use of neonatal cells with ionic properties that change rapidly during development and have thus been poorly characterized and modeled to date. Recently, Kirkton and Bursac demonstrated the ability to create biosynthetic excitable tissues from genetically engineered and immortalized HEK293 cells with well-characterized electrical properties and the ability to propagate action potentials. In this study, we developed and validated a computational model of these excitable HEK293 cells  using existing electrophysiological data and a genetic search algorithm. In order to reproduce not only the mean but also the variability of experimental observations, we examined what sources of variation were required in the computational model. Random cell-to-cell and inter-monolayer variation in both ionic conductances and tissue conductivity was necessary to explain the experimentally observed variability in action potential shape and macroscopic conduction, and the spatial organization of cell-to-cell conductance variation was found to not impact macroscopic behavior; the resulting model accurately reproduces both normal and drug-modified conduction behavior. The development of a computational Ex293 cell and tissue model provides a novel framework to perform paired computational-experimental studies to study normal and abnormal conduction in multidimensional excitable tissue, and the methodology of modeling variation can be applied to models of any excitable cell. in vitro and using computational models to elucidate the mechanisms behind experimental findings. However, tissue culture techniques are limited to working with neonatal, rather than adult, cells, and computational modeling of these cells has proven challenging. In this work, we have a developed a new approach for conducting paired experimental and computational studies by using a cell line engineered with the minimum machinery for excitability, and a computational model derived and validated directly from this cell line. In order to create a model that reproduces the diversity, rather than simply the average behavior, of experimental studies, we have incorporated a simple yet novel method of inherent variability, and explored what types of experimental variation must be incorporated into the model to recapitulate experimental findings. Using this new platform for paired experimental-computational studies with inherent variability, we will be able to study and better understand how changes in cardiac structure such as fibrosis and heterogeneity lead to conduction slowing, conduction failure, and arrhythmogenesis.One of the major challenges in trying to understand how arrhythmias can form in cardiac tissue is studying how the electrical activity of cardiac cells is affected by their surroundings. Current approaches have focused on studying cardiac cells  Making multisite measurements of the transmembrane potential is technically difficult to perform in situ and hence limited information is available to characterize the cells’ complex response to stimuli and drugs. One approach to studying excitable cells in context is to develop detailed in silico computational models of isolated excitable cells and tissues, and to use these models to infer the behavior of the real cells on which they are based. In most cases, computational membrane models are derived from experimental data obtained using various patch clamp techniques, often performed in different labs, under different conditions and often in cells from different species ; any further increase in extracellular potassium results in a failure to fire and propagate action potentials , the extracellular potassium concentration must be increased by at least 2.2 mM to trigger conduction failure, comparable to the threshold observed experimentally.Finally, because the Io 7.4 mM . In our tentials . This efductance ,32; chanin vivo tissues arise from a smaller number of parent cells that divide and grow to confluence, it is possible that cell-to-cell level variability is spatially organized rather than randomly distributed. The impact of this spatial organization of variation on macroscopic conduction was examined using an idealized scenario with a central region of prolonged APD (due to reduced mean IK1 conductance) and reduced cell-to-cell variation. The distribution of ionic properties in the remainder of the monolayer was adjusted such that the overall distribution of IK1 conductance was not altered.Because monolayers and The addition of the central region of prolonged APD and reduced variance does not significantly affect APD or CV during 1Hz pacing . In addiFibrosis was simulated via the addition of a regular field of non-conductive obstacles , which rThe addition of the central region with reduced variation and prolonged APD to the fibrotic tissue results in no significant change in macroscopic conduction behavior at 1Hz or in behavior at shortened diastolic intervals, beyond those observed in the fibrotic tissue alone . Furtherin vitro over a range of conditions, opening the possibility of using this novel computational/experimental framework to explore mechanisms of conduction failure under a variety of conditions.The goal of this work was the develop a mathematical model that allows for paired computational-experimental studies using a simple, synthetic excitable Ex293 cell, and to examine what types of variability were necessary in the model to replicate experimentally observed behaviors in both normal and abnormal conduction conditions. We modeled individual ionic currents using Hodgkin-Huxley formulations and used a parallelized multiobjective genetic search algorithm to identify unknown model parameters to recreate experimental action potential traces. To capture the variability in the electrical properties of the cultured Ex293 cells, we added random variation to current and tissue properties to replicate the degree of electrical variability observed experimentally, and we validated the resulting model across a series of test conditions. Our model captures both the mean behavior and the experimental variability of the engineered cells Various groups have reported extensively on the presence of endogenous potassium –19,33–35 and wholIn our studies, the membrane model’s free parameters (including current densities of each current and temperature-dependent effects on the endogenous potassium currents) were fitted to match a representative action potential recorded in a tissue cultured monolayer using a microelectrode, by coupling a multiobjective genetic search algorithm –43 with Variability in ionic currents can lead to changes in steady state macroscopic conduction properties, as well changes in dynamic behaviors such as restitution , making In order to recreate previously reported experimental variability in Ex293 behavior, we sought to identify the type and degree of variation that was required in the Ex293 model ,9. We chThe need for multifactorial variation is congruent with analysis of experimental intra-monolayer variability . ExperimWhile we incorporated variation as a normal distribution around the baseline model parameters, others have previously used uniform variation on the range of  and performed a post-hoc screening to identify those models whose action potential properties fall within predefined, experimentally-based inclusion criteria . This apIn order to use computational simulations and derive meaningful conclusions from their results, the underlying model must be validated under conditions beyond a single action potential measured at a regular pacing rate. It has previously been noted that variation in channel conductance can lead to significant variation in restitution behavior  and this2), and the extracellular potassium concentration ([K+]o) affect conduction in simulated Ex293 monolayers compared to experimental observations. The simulated response of the Ex293 model to TTX and BaCl2 was qualitatively similar to that in experimental monolayers , barium chloride . HoweveOur model incorporates only two of the endogenous currents that have been identified in HEK293 cells—the endogenous potassium and sodium currents. Chloride currents were excluded from the model because they are poorly characterized under physiological conditions, and there remains significant uncertainty as to their rectification behavior, calcium dependence, and peak density. In addition, we chose not to include endogenous calcium currents because of their relatively small magnitude compared to the other currents . The volWhile the degree of channel conductance variation is likely different for each channel type, we considered a single degree of variation for all channels because of limited information into each channel’s variability. In addition, our model does not incorporate cell-to-cell variability in tissue conductivity. While the variations used in this work were able to reproduce the variability seen in experimental results, the degrees of inter-monolayer variation in different experimental set will need to be modified to match specific experimental variability due different culture conditions, monolayer seeding and handling.Cm is the membrane capacitance, Vm is the transmembrane voltage, Istim is the externally applied stimulus current, and Iionic is the sum of the individual ionic currents  that contribute to the action potential:The excitable cell membrane of a single cell is modeled by the following differential equation:K1 current was modeled with a single activation gate, n, with fast and slow components , and a dependence on extracellular potassium concentration:ext] is the extracellular concentration of potassium. The steady state values of the activation gate (n∞) was described as a function of membrane potential by a Boltzmann sigmoid curve, 1/2 is the voltage at which n∞ = 0.5, k is the slope factor, and nres is the residual value of the gating parameter, independent of membrane potential. Parameters for the Boltzmann equation were determined by fitting experimental data from Kirkton and Bursac [test − EK), where EK is the potassium reversal potential, and normalized before fitting to the Boltzmann curve.The Id Bursac , obtaineVm − EK) and then normalized across all traces. Each resulting conductance trace for test potential v was then fit to the equation:n∞ is the previously determined value of the activation gate at the test potential, τn1 and τn2 are the unknown fast and slow activation time constants, respectively. Fitted values of τn1 and τn2 were then used to formulate a functional definition of the time constants as a function of membrane potential.Time constants of activation as well as the relative contribution of the fast and slow activation components were determine by fitting experimental voltage traces following step voltage change from a holding potential of -40 mV. Voltage traces were converted to conductance traces by dividing by the driving force , they differ in their rate of inactivation, τh1 and τh2. The steady state inactivation curve (h∞) was described by a Boltzmann sigmoid curve, fit to data from Kirkton and Bursac [Supplementary Text 1, the steady state activation curve, as well as activation and inactivation time constants were determined by fitting of experimental sodium current recordings following depolarization from a holding potential of -100 mV. The recordings were converted to conductance traces and normalized to the overall maximum conductance across all test potentials; the resulting traces were then each fitted to the equationh∞ is the value of the previously defined sodium inactivation gate, and m∞ was allowed to be a fitted parameter along with the time constants of activation (τm) and inactivation (τh1 and τh2) as well as the relative contribution of the two inactivation components (d). The resulting steady state activation curve was normalized across voltages and based on its shape, fitted to the form of the sum of two Boltzmann sigmoid curves. The inactivation time constants measured for test potentials above -45 mV were combined with time constants from recovery from inactivation to define the functional definitions of the inactivation time constant.The Id et al. :INa = Gd Bursac , obtaine10 = 2.79, as calculated by Ten Tusscher et al. [1/2 of the sodium steady state activation and inactivation curves, respectively, were determined using data from Nagatoma and by assuming linearity in temperature dependence.Time constants were temperature-adjusted using a Qr et al.  using dar et al. . RightwaNa,wt current was modeled based on the Hodgkin and Huxley formulation with three identical activation gates and one inactivation gate.The I10 of approximately 3 based on temperature dependence data of Nav1.7 channels from Han et al. [Because of limited experimental data on the endogenous channel in Kirkton and Bursac , the sten et al. ,K, wt current was modeled using the form of a slow delayed rectifier current from Ten Tusscher et al. [The Ir et al. , with twThe steady state activation curve and activation time constant were determined by fitting to activation data at 22°C from Yu and Kerchner . BecauseNumerical Methods), and computed the values of the two error functions: the root mean squared error (RMSE) between an experimental AP recording and the simulated AP, and the absolute difference between the experimental and simulated CV. 2-D simulation was performed in a 140 node x 140 node monolayer (dx = dy = 50 micron) with no-flux boundary conditions. The tissue domain was paced at 1Hz at one corner and the action potential tracing of the third action potential from a node 0.6 cm diagonally from the stimulus site was recorded. CV was determined from activation times (50% AP amplitude) at nodes 0.2 cm and 0.8 cm from the stimulus site. RMSE was calculated by aligning the experimental action potential  and simThe multi-objective genetic algorithm was configured to run in parallel across 32 cores using MATLAB’s Parallel Computing Toolbox and Global Optimization Toolbox , with he80  were calculated, as previously described [In order to compare macroscopic conduction properties of simulation results with those obtained experimentally using a voltage sensitive dye and an optical fiber recording array, averaging of local simulated membrane potentials in small circular regions was performed to simulate optical recording of model results. While the experimental optical fiber array has fibers with diameter of 750 μm arranged in a 20-mm diameter hexagonal bundle, spacing between the optical fiber array and the tissue sample results in a wider effective field of view for each fiber. As a result, while our simulated optical sensors were spaced with 750 μm center-to-center spacing, each sensor averaged potentials over a circular region of diameter 1100 μm. The resulting voltage traces for each of the 504 simulated optical sensors was analyzed using custom MATLAB software developed for analysis of experimental optical mapping recordings, and the CV and APDInter-monolayer variation of conductances and conductivity was generated by selecting scaling factors for each monolayer from random normal distributions with mean 1 and specified standard deviation. Base model parameters were then scaled by these factors and the new monolayer-specific mean parameters were provided to the Cardiowave simulator. Cell-to-cell variability was incorporated directly into the membrane model. Each node’s channel conductance is randomly selected at the start of the simulation from a random normal distribution with the selected mean monolayer channel conductance and a specified standard deviation see . Simulatm dt-1), resting membrane potential, APD and action potential amplitude.The sensitivity of the membrane model to perturbation was assessed by independently altering the conductance of each current in a range from 50% to 150%, and measuring the impact on conduction properties. Sensitivity was measured in a simulated strand of tissue . In addition to the macroscopic conduction properties measured using simulated optical sensors, several addition properties were measured using the action potential traces from the center-point of the strand, analogous to experimental sharp electrode recording. These properties included the maximum upstroke velocity . The standard (S1-S2) protocol was used wherein the strand was stimulated from one end at 2 Hz for 10 pulses (S1) followed by a premature stimulus of the same amplitude (S2), and the CV and APD resulting from the S2 stimulus were recorded. The S1-S2 interval was decreased until the S2 pulse no longer elicited an action potential.Na and INa,wt currents. Blockade of the IK1 channel due to BaCl2 was simulated by scaling the IK1 conductance based on the degree of block at -100 mV and the membrane potential  was simulated by simultaneously altering the conductance of both the Iotential . The effConduction was simulated in a 2D continuous monodomain model . Non-conductive fibrosis-like obstacles were added to the tissue domain, with each obstacle 100 μm x 100 μm in size and with 100 μm spacing between obstacles. Obstacles were decoupled from neighboring tissue nodes to establish no-flux boundary conditions. A central region of homogeneity with a diameter of 140 μm (70% of strand width) was established. Within this region, the mean potassium conductance was one standard deviation (12.5%) below the monolayer mean, and the standard deviation of cell-to-cell variation was narrowed to 0.0625 for all ionic properties to create relative homogeneity with preserved but reduced cell-to-cell variability. Cell-to-cell ionic variation in the remainder of the tissue was resampled in order to preserve the distribution of cell-to-cell variation across the full tissue. The central region remained fully coupled to the surrounding tissue. The previously described standard S1-S2 protocol was used to assess restitution behavior.All simulations were performed using the Cardiowave software package , a cardiAll data is presented as mean +/- standard deviation unless otherwise specified. Comparison of model and experimental variability was performed using Levene’s test for equality of variances with an alpha value of 0.05. Comparison of macroscopic CV, APD and failure behavior in the presence of fibrosis and spatial organization of variability was performed using a two-way ANOVA with two between-subjects measures (fibrosis and organization). Comparison of restitution behavior was performed using three-way ANOVA with one within-subjects measure  and two between-subjects measures (fibrosis and organization), and post-hoc pairwise comparison was performed using Fisher’s LSD.S1 Text(PDF)Click here for additional data file.S1 Fig(A) Experimentally recorded currents during “AP-clamp”. Note that the duration of the experimental action potential used to generate this recording is longer than the mean duration which was matched by the model. (B) Action potential trace used for simulated AP clamp (C) Model currents at 23C qualitatively matches experimental currents.  Model currents at 35C. Temperature induced variation in conductances and activation/inactivation properties lead to a much larger inward sodium current.(PDF)Click here for additional data file.S2 FigStandard deviations of measured single cell APD and upstroke velocity were obtained for each experimental monolayer (n = 6). Monolayers exhibit relatively little APD variability but substantially more maximal upstroke velocity variability within each monolayer. The standard deviation of each parameter between monolayers is indicated by the dashed red line. The degree of cell-to-cell upstroke velocity variation within individual monolayers is comparable to the variation seen between different monolayers, while cell-to-cell variation of action potential duration within individual monolayers is 3-5x smaller than that seen between different monolayers. Models that include only inter-monolayer variability and not cell-to-cell variability fail to reproduce the experimentally observed variability of upstroke velocity within individual monolayers.(PDF)Click here for additional data file.S3 FigNa and IK led to changes in both CV and APD while variation of the endogenous currents affected APD without affecting CV. Variation in both INa and IK,wt led to changes in action potential amplitude, while only INa variation affected the maximal upstroke velocity.Current densities of each of the four constitutive currents were independently varied from 50% to 150% and the effect of on conduction and action potential shape properties was measured. Variation in I(PDF)Click here for additional data file.S4 FigIK1 block due to barium chloride was modeled as dependent on membrane potential and degree of block at -100 was used as a substitute for drug dose.(PDF)Click here for additional data file.S5 FigThe model is able to replicate experimental variability in several isolated single cell properties other than those used to set levels of conductance variation.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


One eye required vitrectomy during follow-up. Recurrent NVD was observed in 2 eyes, which resolved after repeated injections. The remaining 12 eyes were stable over a mean follow-up period of 8.3 months. The mean area of the NVD in 14 patients without vitrectomy was 0.22 ± 0.11 mm2 (p = 0.000) at the last visit. Intravitreal conbercept injections combined with intensive PRP are an effective and safe treatment for PDR with NVD. Quantitative information on NVD can be obtained with OCTA, which may be clinically useful in evaluating the therapeutic effect.This study reports the short-term efficacy and safety of intravitreal conbercept injections for neovascularization at the disc (NVD) in patients with proliferative diabetic retinopathy (PDR). Conbercept is a recombinant fusion protein with a high affinity for all isoforms of vascular endothelial growth factor (VEGF)-A, placental growth factor and VEGF-B. A prospective case series study was conducted in 15 patients (15 eyes). Patients had complete ocular examinations and received a 0.5 mg intravitreal conbercept injection followed by supplemental pan-retinal photocoagulation (PRP). Optical coherence tomography angiography (OCTA) was performed before and after treatment. Before treatment, the mean NVD area was 1.05 ± 0.33 mm Proliferative diabetic retinopathy (PDR) is a severe stage of DR and one form of vision-threatening DR. Pan-retinal photocoagulation (PRP) is the established treatment for PDR, as it is effective and has preserved vision in numerous patients over the past several decades4.Diabetes mellitus (DM) is one of the world’s fastest growing chronic diseases, and diabetic retinopathy (DR) is a specific microvascular complication of DM. As the prevalence of DM continues to increase, DR is still the leading cause of acquired vision loss worldwide in middle-aged and therefore economically active people9. Conbercept is a recombinant fusion protein that consists of the VEGF binding domains of the human VEGFR-1 and VEGFR-2 combined with the Fc portion of the human immunoglobulin G1. In addition to having a high affinity for all isoforms of VEGF-A, it also binds to placental growth factor (PlGF) and VEGF-B10. As the use of conbercept in the treatment of PDR is off-label, there are few reports concerning this topic. We have conducted a prospective study on the short-term efficacy and safety of intravitreal conbercept injections combined with laser treatment for neovascularization at the disc (NVD) in patients with PDR.As vascular endothelial growth factor (VEGF) plays a role in the process of neovascularization in PDR, anti-VEGF agents including ranibizumab and bevacizumab have been assessed for the treatment of PDR in recent years, and the results were satisfactory12. To obtain quantitative information on NVD, OCTA was performed before and after treatment, and the NVD area was calculated.The introduction of optical coherence tomography angiography (OCTA) into clinical practice allows the detection and visualization of the blood flow and morphology of retinal vessels and is useful to monitor different NVD subtypes, their development, and the efficacy of treatment regimens as well as to define new vessel morphological detailsThis prospective case series included 15 eyes from 15 patients with diabetes who presented at the Peking Union Medical College Hospital between January and September 2016. They had been clinically diagnosed with PDR with active NVD. All the patients were informed about the purpose and treatment procedure, and written informed consent were obtained before intravitreal injections. All procedures conformed to the tenets of the Declaration of Helsinki. The methods were carried out in accordance with the approved guidelines, and the study was approved by the Ethics Committee of the Peking Union Medical College Hospital.Inclusion criteria included patients with PDR with disc neovascularization, patients with clear media that allowed fundus examinations and OCT angiography examinations, and patients who had not been treated with intravitreal anti-VEGF or steroid injections within six months. Exclusion criteria included patients with opaque media that influenced fundus examination, including corneal opacity, cataracts and vitreous hemorrhages; previous eye diseases such as glaucoma and retinal diseases besides DR, such as retinal vein occlusion or AMD; eye infections and intraocular inflammations; tractional retinal detachment; and penetrating ocular trauma or intraocular surgery within the previous 6 months prior to intravitreal injection. Patients with uncontrolled hypertension, recent myocardial infarction or cerebral vascular incidents were also excluded.All patients had a complete ocular examination before and after treatment that included the following measurements: best corrected visual acuity (BCVA) using the International Standard Visual Acuity Chart, intraocular pressure (IOP) using a non-contact tonometer, ocular motility, pupillary light reflex, slit-lamp examination, fundus examination using indirect ophthalmoscope, fundus photography , spectral domain optic coherence tomography, and OCT angiography .2. As OCT angiography produces multiple scans, the best image for the detection of new vessel architecture was selected for use in the study. The B-scan OCT was used to confirm the presence of NVD, and new vessels were observed as preretinal proliferative structures or protrusions into the vitreous cavity with positive flow signals. Segmentation of the inner border of the B-scan was manually moved to the vitreous cavity, just above the new vessels, and the outer border was adjusted to minimize the depiction of the superficial vascular plexus13.AngioVue disc examinations were performed by one experienced eye specialist (ZLJ). The scanning pattern was 4.5 × 4.5 mmA circle with a 3-mm diameter was drawn with its center at the optic disc, and the structure of the NVD was outlined with white lines using Adobe Photoshop (CS6), with the background colored black. The processed image was saved as a black and white two-color chart. If mentclass2pt{minimAll patients received an explanation of the off-label use of conbercept in PDR and its potential benefits and risks. Informed consents were obtained, and a prophylactic topical antibiotic (Levofloxacin) was instilled for 3 days before treatment.After topical anesthesia and sterilization of the operating field, 0.5 mg/0.05 mL of conbercept was injected intravitreally at the inferotemporal pars plana (4 mm posterior to the limbus). After the injection, the intraocular pressure was measured, and prophylactic topical antibiotic drops were instilled for 3 days. Intensive retinal laser photocoagulation was applied beginning two weeks after the intravitreal injection. A slit-lamp laser was used, and patients received laser treatment under topical anesthesia. For patients without previous PRP, laser therapy was completed after three to four sessions, with a total of 1600 to 1800 burns. For patients with previous PRP, laser treatment was applied where the former laser treatment was not sufficient.SPSS statistical software version 20.0  was used for statistical analysis. A paired-samples t-test was used to compare the area of the NVD before treatment and after intravitreal conbercept injection. A probability (P) value < 0.05 was considered statistically significant.A total of 15 patients (15 eyes) with type 2 diabetes mellitus were investigated in this study. Of the 15 patients, 7 were male and 8 were female. The mean age was 42.9 ± 10.9 years (range 29–64 years), and the demographic details are shown in Table In fundus biomicroscopy or color photography, new vessels of the optic disc usually manifest as irregular blood vessels on the optic nerve head or protruding into the vitreous cavity; however, the detailed structures of the NVD are sometimes not clear and simply manifest as redness of the optic papilla or an unclear optic disc border. During FA examination, the neovascularization of the disc is clear for only a few seconds, and then the flowing dye leakage makes it difficult to visualize the vessel profile. During OCT angiography, we were able to observe the detailed structures of the NVD clearly. In the 15 eyes, most of the NVD manifested as an irregular proliferation of fine vessels on or just near the optic disc . The time to the first visit after the initial intravitreal conbercept therapy ranged from 6 to 9 days (mean 7.5 days). The mean area of the NVD decreased to 0.56 ± 0.17 mm2 (range 0.25–0.83 mm2)  at the last visit (Table The mean follow-up period was 8.3 months range: 6–14 months). The mean area of the NVD in 14 patients without vitrectomy was 0.22 ± 0.11 mm4 months.14. According to the World Health Organization (WHO), it is estimated that DR accounts for 4.8% of the cases of blindness (37 million) worldwide15.Diabetic retinopathy remains a major cause of visual impairment and blindness in the world. Over one-third of the world’s 285 million people with DM are estimated to have DR, and one third of these (approximately 31.7 million) have vision-threatening diabetic retinopathy (VTDR)4. However, this is not always the case, as approximately 60% of patients with PDR who respond to scatter laser treatment show regression of neovascularization within 3 months, and many require additional laser treatments17. Ten out of 15 eyes in our study had previous PRP treatment, but NVD was still active, and further treatment was needed. Neovascularization on and around the optic disc and vitreous hemorrhages were found to be more frequently associated with severe visual loss despite PRP in the Diabetic Retinopathy Study (DRS) and ETDRS7. Meanwhile, PRP is impossible to apply completely in patients with media opacities such as vitreous hemorrhages or cataracts. It has been reported that despite frequent follow-up examinations and timely PRP, the 5-year cumulative rate of pars plana vitrectomy in ETDRS patients is 5.3%17. Therefore, the need exists for an adjunctive therapy for PDR.Laser photocoagulation is the traditional treatment for PDR, and PRP has been effective at preserving vision in numerous PDR patients over the past several decades. According to the Diabetic Retinopathy Study, PRP reduced the risk of severe visual loss by more than 50%8. Avery et al. studied the angiographic leakage from abnormal new vessels after intravitreal bevacizumab injections and showed that all patients with neovascularizations seen with fluorescein angiography (44 eyes) had an at least partial or complete reduction of leakage from the neovascularization within 1 week after the injection5. Minnella et al. also reported regression and reduction of the area of new vessels as early as 1 month after intravitreal injection in all studied eyes and a clearing of the bleeding in eyes with recurrent vitreous hemorrhages. These effects were maintained for 3 months in all eyes and tended to be stable at 9 months with no side effects from the injections18. In the series reported by Ababneh et al., more than 50% of the new vessels were stable or improved at 6 months6. Arevalo et al.19 and Arevalo and Garcia-Amaris7 found that 27 eyes (61.4%) with retinal neovascularization completely regressed after intravitreal bevacizumab injections, and 15 eyes (34.1%) had a partial regression of retinal neovascularization seen via fundus examination and fluorescein angiography, whereas 2 eyes of 2 patients (4.5%) had no regression of retinal neovascularization. Yang and colleagues observed 20 eyes from 17 patients with high-risk PDR and showed that the combination of intravitreal bevacizumab and PRP led to the rapid clearance of VH, regression of retinal neovascularization, and visual improvements in the treatment of high-risk PDR20.As VEGF plays a role in the process of neovascularization in many ocular vascular diseases, anti-VEGF agents have been frequently used for the treatment of PDR in recent years. Bevacizumab was the first anti-VEGF agent used for PDR. It is reported to be a well-tolerated medication that causes regression of abnormal diabetic neovascularizations and a rapid resolution of vitreous hemorrhages9.The DRCR Network developed Protocol S to compare intravitreal ranibizumab with PRP for the treatment of PDR in 394 eyes. At 2 years, the visual outcome of the ranibizumab-treated eyes was non-inferior to the PRP-treated eyes, with a mean visual acuity difference of +2.2 letters in favor of the ranibizumab group. The results of this trial suggest ranibizumab injections are a safe and effective alternative to PRP for the management of PDR21. It was approved to treat neovascular AMD by the China State FDA in December 2013. Compared with aflibercept, conbercept is larger and contains the fourth binding domain of VEGFR-2. It has a lower VEGF dissociation rate, higher binding affinity, decreased adhesion to the extracellular matrix and lower isoelectric point, which results in a longer clearance time22. Relatively few studies have been performed on the intravitreal injection of conbercept for the treatment of neovascularization in PDR. Some studies have focused on preoperative conbercept pretreatments and found that it could be an effective adjunct to vitrectomy by reducing the chances of intraoperative bleeding, accelerating postoperative vitreous clear-up and helping stabilize visual acuity restoration for PDR24.Conbercept is a soluble receptor decoy that blocks all isoforms of VEGF-A and VEGF-B as well as PlGF, which has a high binding affinity for VEGF and a long half-life in the vitreous bodyIn our study, 15 PDR patients (15 eyes) with active NVD were treated with intravitreal conbercept injections (0.5 mg) followed by laser treatment at least 2 weeks after injection. All NVD regressed to different degrees, over a mean period of 7.5 days, manifesting as a significant decrease in the mean NVD area. This showed that conbercept can quickly cause regression of NVD. However, one eye required vitrectomy surgery during follow-up due to a dense, persistent vitreous hemorrhage and proliferative membrane formation. The recurrence of NVD was observed in two eyes due to insufficient laser photocoagulation, and NVD regressed after repeated conbercept injections. At the last visit, at a mean time of 8.3 months, all eyes had anatomic (determined by OCTA) improvements and visual acuity stabilization, demonstrating that the combination of intravitreal conbercept injections and laser photocoagulation is a safe and effective treatment for PDR. In addition, we found that the fibrous membranes on the disc were removed easily with little hemorrhaging during the surgery.et al.19 and Arevalo and Garcia-Amaris7 found that 21 eyes (47.7%) needed a second injection of bevacizumab, and 7 eyes (15.9%) needed a third injection due to the recurrence of neovascularization. In Yang’s study, recurrent retinal neovascularization with minor preretinal hemorrhaging was observed in 6 eyes (30%) 3 months after the first injection, which resolved after repeated bevacizumab injections20. In the current study, the NVD of 2 eyes (13.3%) recurred after the initial conbercept treatment and was stable after a second injection combined with laser photocoagulation. Kubota and associates demonstrated that vascular endothelial cells with decreased expression of VEGF were still present in the proliferative tissues after a bevacizumab injection. Anti-VEGF therapy temporally reduces the blood flow of the new vessels; however, it does not induce the complete regression of the vascular endothelial cells in new vessels25. We consider PRP to play an important role in preventing the recurrence of neovascularization.The recurrence of neovascularization is an important concern in anti-VEGF treatments in PDR. Arevalo 20. In the current study, one eye had proliferative membrane formation without obvious tractional retinal detachment after conbercept injection. We reason that preexisting fibrovascular tissue contractions and vitreoretinal adhesions may be risk factors in tractional retinal detachment, and attention should be drawn before anti-VEGF treatment in this kind of PDR patient.Tractional retinal detachment after injection is another concern of anti-VEGF treatments. Theoretically, the anti-VEGF pharmacologic agent has fibrosis-inducing abilities and increases the probability of tractional retinal detachment. In the literature, tractional retinal detachment was reported in some PDR cases after intravitreal bevacizumab26. Using OCTA, the number, course, size and extension of NVD can be determined, but these features cannot be properly assessed using FA26. Because of the limits of the extent of OCTA scanning, not all neovascularizations could be quantitatively evaluated, so we chose NVD as the study metric. In our current study, the vascular structures of the NVD could be visualized clearly on OCT angiograms, even with a thin preretinal hemorrhage, and the area of the NVD could be calculated for statistical analysis. Another limit of OCTA is that it cannot show leakage of the neovascularizations, so it is not able to assess the activity of the NVD directly. In a recent study, Ishibazawa and colleagues have put forward the concept of exuberant vascular proliferation (EVP), which is the intense growth of irregular small-caliber vessels located at the margin of new vessels, likely representing active proliferation13. In our study, most of the patients did not have FA, so we were not able to compare OCTA results to the leakage in FA. However, EVP was present in 86.7% (13/15) of patients before treatment, and all these vessels converted to pruned vessels with residual, larger vascular loops approximately 1 week after conbercept injection, manifesting as the disappearance of EVP in OCTA13. While the concept of EVP is the assessment of the activity of the NVD qualitatively, we used NVD areas to compare changes in the NVD before and after treatment quantitatively. We noticed that all of the EVPs disappeared 1 week later, but NVD areas continued to decrease from 0.56 ± 0.17 mm2 to 0.22 ± 0.11 mm2 at last visit.As we decided to quantitatively evaluate changes in abnormal vessels in PDR after treatment, OCTA was chosen for the measurements of the area of neovascularization. The introduction of OCTA into the clinical practice is useful to monitor different NVD subtypes, their development, and the efficacy of treatment regimens as well as to define new vessel morphological details without the need for dye injections and to avoid dye leakageThe limitations of this study include the fact that it had a relatively small number of patients and a short-term follow-up period. Meanwhile, there was no control group of NVD in PDR patients without intravitreal conbercept treatment. In addition, we hope that an automatic quantitative method to evaluate NVD and wide-angle OCTA to evaluate neovascularizations at different locations will become available in the future.In conclusion, conbercept is a well-tolerated medication that causes a quick regression of NVD, and intravitreal conbercept injections combined with intensive PRP is an effective treatment for NVD in PDR patients. Quantitative information on NVD can be obtained with OCTA, which may be clinically useful in evaluating the therapeutic effect of treatments for NVD in PDR. A prospective study with long-term follow-up is needed to confirm the therapeutic benefits of conbercept and to evaluate ocular and systemic adverse effects.


Standard two-dimensional (2D) cell migration assays do not provide information about vertical invasion processes, which are critical for melanoma progression. We provide information about three-dimensional (3D) melanoma cell migration, proliferation and invasion in a 3D melanoma skin equivalent (MSE) model. In particular, we pay careful attention to compare the structure of the tissues in the MSE with similarly-prepared 3D human skin equivalent (HSE) models. The HSE model is identically prepared to the MSE model except that melanoma cells are omitted. Using the MSE model, we examine melanoma migration, proliferation and invasion from two different human melanoma cell lines. One cell line, WM35, is associated with the early phase of the disease where spreading is thought to be confined to the epidermis. The other cell line, SK-MEL-28, is associated with the later phase of the disease where spreading into the dermis is expected.3D MSE and HSE models are constructed using human de-epidermised dermis (DED) prepared from skin tissue. Primary fibroblasts and primary keratinocytes are used in the MSE and HSE models to ensure the formation of a stratified epidermis, with a well-defined basement membrane. Radial spreading of cells across the surface of the HSE and MSE models is observed. Vertical invasion of melanoma cells downward through the skin is observed and measured using immunohistochemistry. All measurements of invasion are made at day 0, 9, 15 and 20, providing detailed time course data.in vivo, with a well-defined stratification of the epidermis that is separated from the dermis by a basement membrane. In the HSE and MSE we find fibroblast cells confined to the dermis, and differentiated keratinocytes in the epidermis. In the MSE, melanoma cells form colonies in the epidermis during the early part of the experiment. In the later stage of the experiment, the melanoma cells in the MSE invade deeper into the tissues. Interestingly, both the WM35 and SK-MEL-28 melanoma cells lead to a breakdown of the basement membrane and eventually enter the dermis. However, these two cell lines invade at different rates, with the SK-MEL-28 melanoma cells invading faster than the WM35 cells.Both HSE and MSE models are similar to native skin The MSE and HSE models are a reliable platform for studying melanoma invasion in a 3D tissue that is similar to native human skin. Interestingly, we find that the WM35 cell line, that is thought to be associated with radial spreading only, is able to invade into the dermis. The vertical invasion of melanoma cells into the dermal region appears to be associated with a localised disruption of the basement membrane. Presenting our results in terms of time course data, along with images and quantitative measurements of the depth of invasion extends previous 3D work that has often been reported without these details. Melanoma is a deadly form of skin cancer  that is Previous studies about the spreading of melanoma have focused on examining the spatial extent of population expansion, cell migration, cell proliferation, cell-to-cell adhesion and protein-expression on two-dimensional (2D) surfaces . These 2in vivo  models . These 3Two melanoma cell lines, WM35 (RGP)  and SK-MQueensland University of Technology (QUT) human research ethics provides written approval for the skin samples to be used in this study . The samples come from patients undergoing abdominoplasty surgery and breast reduction surgery .2 and 95% air.Human keratinocyte cells are isolated from skin and cultured in full Green’s medium following protocols described in 2 and 95% air.Human fibroblast cells are isolated following protocols in 2 and 95% air.The human melanoma cell lines, WM35 and SK-MEL-28 are cultured as described in Both melanoma cell lines, WM35 and SK-MEL-28, are validated using short tandem repeat profiling . This means that the cell lines that we use are identical to the reference samples held in Cell Bank.4) and primary fibroblast cells (1 × 104) are seeded onto the DEDs in full Green’s medium and incubated at 37 °C, in 5% CO2 and 95% air for 2 days. Subsequently, the DEDs with cells, from now onwards referred to as HSE, is submerged in full Green’s medium for 2 days. These HSEs are then cultured at an air-liquid interface on sterile stainless steel grids with full Green’s medium for 9, 15 and 20 days. HSE is also collected at day 0, just before the DED is lifted to the air-liquid interface, as a reference sample.HSE models are established using the skin collected from donors undergoing elective plastic surgery. The protocol for establishing the HSE model is given in 3) or SK-MEL-28 (5 × 103), in addition to primary keratinocyte (2 × 104) and primary fibroblast (1 × 104) cells on the individual DEDs. This protocol of adding all the cells together on DEDs is standard in all the previous DED studies summarised in To construct the MSE models, we follow the same protocol for the HSE model, and include melanoma cells, WM35  fitted with a Nikon digital camera.An MTT -2,5-diphenyltetrazolium bromide) (Thermo Scientific) assay is performed to check the viability of cells in the HSE and MSE models. HSE and MSEs collected on day 0, 9, 15 and 20 are submerged in 0.5 mg/ml w/v MTT solution and incubated at 37 °C, in 5% COHaemotoxylin and eosin (H&E) staining is used to characterise the tissue structure in the HSE and MSE models. MTT stained HSE and MSEs are fixed using 10% neutral buffered formalin , processed in an automated vacuum tissue processor  and embedded in paraffin wax. All samples are sectioned to 5 µm thickness using a microtome . All HSE and MSE samples are first visually examined to see the spatial extent of the MTT positive region. Then, each sample is divided using a sterile blade, through the centre of the MTT positive region. The two smaller samples of tissue are each embedded in paraffin wax. These smaller samples are then further sectioned into 5 µm thick tissue sections using a microtome. This procedure allows us to explore the depth of vertical invasion that is close to the centre of where the population of cells is initially placed in a circular barrier onto the DED. Furthermore, by examining the depth of vertical invasion in the various 5 µm thick sections, we can examine whether the depth of vertical invasion depends on the lateral position. In summary, we find that the patterns of vertical invasion appear to be independent of the lateral position. Overall, in each experiment, we examine approximately 80–120 sections that are 5 µm in thickness. This means that we examine the vertical invasion of melanoma cells within a region extending from the centre of the initial population to approximately 400–600 µm away from that centre.Sections are first deparaffinised in 100% xylene and rehydrated in graded ethanol series of 100%, 90% and 70%, and followed by distilled water. These sections are incubated in Harris haematoxylin  followed by differentiation with 1% acid alcohol, bluing with Scott’s tap water solution and counterstaining with alcoholic eosin (HD Scientific). H&E stained sections are dehydrated in 90% and 100% ethanol, cleared with 100% xylene and mounted on coverslips using Pertex® mounting medium . All stained sections are imaged using an Olympus BX41 microscope fitted with an Olympus digital camera .Immunohistochemistry is performed on the paraffin-embedded (5 µm) sections. Paraffin embedded sections are deparaffinised and rehydrated as previously described in We use ImageJ  to measuWe first outline the MTT assay performed on both the HSE and MSE models. Results of the MTT assay, shown in An interesting result detected by the MTT assay is the formation of visually prominent colonies of cells in the central region of the MSE for the SK-MEL-28 cell line at day 0, as shown in the inset of in vivo. To investigate this, we perform histological analysis and describe our results in this section, ‘Proliferation, Migration and Invasion of Melanoma Cells on the MSE Model’. However, we also use immunohistochemistry to examine the spatial and temporal distribution of markers for cell migration, cell proliferation and cell invasion in ‘Quantification of Melanoma Invasion’. Since the main focus of this work is about cell migration, cell proliferation and cell invasion, we choose to present all histological analysis about tissue structure in the The next aim in our study is to examine the tissue structure of the HSE and MSE models, and to compare the structure of the tissue in these models with the structure of native human skin in vivo. In particular, we see the formation of distinct epidermal and dermal regions, Cross-sections through the HSE and MSE models are generated for H&E staining. Results at day 9, 15 and 20, showing HSE and MSE cross-sections, reveal morphological similarities to native human skin in vivo (The basement membrane separates the epidermal and dermal compartments, and is a prominent feature of native human skin in vivo . The basin vivo . The posin vivo by disturbing the basement membrane (in vivo stages of melanoma progression in vitro.We observe a weakly stained, mostly-continuous basement membrane in skin models constructed using WM35 cell lines at day 9, as shown in membrane . Therefoin vitro are similar to native human skin in vivo. The marker loricrin identifies terminally differentiating cells in the epidermis (Lastly, positive staining of the terminally differentiating epidermis confirms that both the HSE and MSE models constructed pidermis . Therefopidermis . Hence, in vitro HSE model is consistent with native human skin in vivo. In contrast, the MSE models do not always have a well-defined basement membrane. At early times in the experiments we see that the basement membrane is formed and present in the MSE model. However, at later times, the basement membrane in the MSE model is partially absent. These differences between the MSE and HSE models suggest that the presence of melanoma cells in the MSE models might lead to disruptions in the basement membrane. Furthermore, we hypothesise that this disruption is associated with vertical invasion.In summary, the loricrin staining suggests that the physiology of the HSE and MSE models is consistent with native skin. Furthermore, our findings show that HSE models have well-defined stratified epidermal and dermal regions that are separated by a basement membrane. This confirms that the Certain key features of cancer progression, including melanoma, are thought to be the proliferation, migration and invasion of cancer cells . TherefoThe MTT assay provides information about the radial spreading of cells across the MSE model. In addition to radial spreading, we also aim to observe and quantify the vertical invasion of melanoma cells, and in particular we wish to focus on cell lines that are associated with both the early and later stages of melanoma progression. RGP melanoma is generally associated with melanoma cells confined to the epidermal region of the skin . PreviouTo make this comparison we examine data from the MSE with the WM35 melanoma cell line in in vivo (Migrating cells in the MSE models are detected using the marker vimentin . In thisin vivo . Since fin vivo . Note thin vivo .Vertical invasion of melanoma cells into the MSEs is detected by the marker S100. Invasion in this context refers to vertical spreading of melanoma cells into the dermis. Our previous studies show that S100 is a reliable marker that identifies both the WM35 and the SK-MEL-28 melanoma cell lines . Since tComparing the size of the melanoma colonies over time in both MSE models shows that the colonies of SK-MEL-28 melanoma cells are larger than the colonies of WM35 melanoma cells. These differences are most evident at day 20, as shown by comparing the images in To explore whether the differences in invasion of the two melanoma cell lines might be associated with any difference in cell size, we measure the size of WM35 and SK-MEL-28 cells, as shown in An interesting result from the MSE with the WM35 melanoma cell line is that we observe the invasion of small colonies of WM35 melanoma cells into the dermis at day 20, as shown in Overall, these results showcase the successful establishment of a reliable and enduring MSE model that can be used to examine the migration, proliferation and invasion of melanoma cells from two different cell lines associated with RGP and metastatic stages of melanoma progression. However, in addition to providing qualitative information about the spatial and temporal distribution of different cell types in the MSE models, we also provide quantitative information about the invasion process.To further examine the differences in the invasion patterns associated with the WM35 and SK-MEL-28 melanoma cell lines, we measure the depth of cell invasion into the dermal region over time. The depth of invasion is taken to be the distance from the top of the dermis to the deepest region invaded by the melanoma cells, as shown in Results in Previous research has measured the invasion of melanoma cells into the dermis . These sIn summary our results suggest that the WM35 and the SK-MEL-28 melanoma cell lines both exhibit invasive properties and have the ability to enter the dermis in our model. This is interesting because the WM35 melanoma cell line is thought to be associated with the early phase of melanoma progression where the cells are confined to the epidermis. We do, however, observe differences in the invasive properties of the two cell lines. For example, the WM35 cells appear to take a long duration of time to enter the dermis than the SK-MEL-28 cell. Our study does not explain why the WM35 cells take a longer period of time to enter the dermis. However, we anticipate that these differences could have many explanations. For example, the disruption of the basement membrane could be driven by some kind of chemical signal, and the differences in the speed of invasion could be associated by differences in the production rates of such chemical signals. Exploring these ideas is a topic for future research. Overall, our qualitative observations and quantitative measurements suggest that the WM35 melanoma cell line is less invasive than the SK-MEL-28 melanoma cell line.in vivo than 2D studies. HSE skin models constructed using DED are used in many research areas including wound healing and burn studies (in vivo it can be adapted to study melanoma proliferation, migration and invasion patterns. Melanoma has various phases of progression and 2D models are limited since 2D models cannot be used to study vertical invasion. In contrast, 3D skin-based melanoma models can be used to study vertical invasion, as well as exploring how melanoma cells interact with surrounding cells and tissues.In summary, 3D skin model studies are more realistic, and more closely resemble native human skin  studies . Since tin vitro MSE model using cell lines from early and late phases of melanoma. The MSE model incorporates ether WM35 melanoma cells or SK-MEL-28 melanoma cells, as well as primary keratinocytes and primary fibroblasts. Our MSE models are capable of examining melanoma progression for up to 20 days, which is the longest time point we have analysed. Collectively, our results suggest that MSE models constructed in vitro have similar tissue structure to native human skin. The melanoma cells in the MSE models proliferate, migrate and invade into the dermis as observed in native human skin in vivo. However, these two cell lines from the RGP and metastatic phase of melanoma lead to different patterns of invasion. Importantly, the MSE models enable quantitative measurements of the invasive process to be made, and allow us to quantitatively compare the progression of the two different cell lines.In this study we develop an BRAF mutations (Lastly, it could be of interest to extend this work by using cell lines associated with the VGP state of the disease in the MSE and comparing these additional results to our current study. Additionally the MSE could also be further developed as a pre-clinical platform to investigate the effects of anti-melanoma drugs. Both melanoma cell lines used in the current study have utations , hence tutations . The alt10.7717/peerj.3754/supp-1Supplemental Information 1Additional resultsClick here for additional data file.


Multiple myeloma patients who are relapsed or refractory to both proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) have been reported to have poor outcomes. Bendamustine has been reported to have an antitumor effect in newly diagnosed as well as relapsed/refractory multiple myeloma (RRMM). The aim of this retrospective study was to evaluate the efficacy of bendamustine therapy in heavily pretreated MM patients who were refractory to PIs and IMiDs.2 to 120 mg/m2 on days 1 and 2 of 28-day cycles.Nineteen RRMM patients treated either with bendamustine and steroids (n=13) or a combination of bendamustine with novel drugs (n=6) were included. The median number of previous treatment lines was 5 (minimum-maximum: 3-8) and median time from diagnosis was 6 years (minimum-maximum: 1-16). All of the patients were resistant to at least one of the IMiDs and one of the PIs. Bendamustine was given at doses ranging from 90 mg/mA median of 2 (minimum-maximum: 1-8) treatment cycles was administered per patient. The toxicity of bendamustine was mild and mostly of hematological origin. No complete remission was achieved. There was partial remission and stable disease in 21% and 11% of the patients, respectively. Sixty-eight percent of patients had progressive disease. The median progression-free survival and overall survival was 2 and 4 months, respectively.Bendamustine therapy was well tolerated but showed limited anti-myeloma activity in heavily pretreated patients who were refractory to IMiDs and PIs. Multiple myeloma (MM) is the second most common hematological malignancy, accounting for an estimated 1% of all cancers ,3. RecenBendamustine is a nitrogen mustard-based alkylating agent shown to be effective in the treatment of various hematologic malignancies. It can be safely administered to patients both with mild to moderate renal insufficiency and moderate hepatic insufficiency ,5.Bendamustine has been used for more than a decade for the treatment of MM, either as the sole therapy or in combination with steroids and other chemotherapeutics including novel agents . ConsideIn this retrospective analysis we tried to explore the real-life effectiveness and safety of bendamustine in heavily pretreated MM patients refractory to IMiDs and PIs.Patients were identified by reviewing the medical records at the Hematology Department of Cerrahpaşa Medical Faculty, İstanbul University. This retrospective study included 19 patients who were RR to at least one of the IMiDs  and one of the PIs (carfilzomib and bortezomib). Patient characteristics before bendamustine treatment are shown in 2 to 120 mg/m2 and it was administered intravenously on days 1 and 2 of a 28-day cycle as per the protocol described in previous studies . However, we did not observe a benefit of bendamustine treatment in patients who were refractory to IMiDs and PIs. It is important to reiterate that our sample size does not permit us to make a precise statement. However, based on our experience with this relatively small number of patients, there is no clear recommendation to be made for the use of bendamustine in IMiD- and PI-resistant heavily pretreated MM patients. Such patients should be encouraged to participate in clinical trials evaluating new approaches.


Preeclampsia (PE) is associated with a high risk of perinatal morbidity and mortality. However, there is no consensus in the definition of high-risk women.To question current definition of high PE risk and propose a definition that considers individual heterogeneity to improves risk classification.A stratified analysis by parity was conducted using the Swedish Birth Register between 2002–2010 including 626.600 pregnancies. The discriminatory accuracy (DA) of traditional definitions of high-risk women was compared with a new definition based on 1) specific combinations of individual variables and 2) a centile cut-off of the probability of PE predicted by a multiple logistic regression model.None of the classical risk-factors alone reached an acceptable DA. In multiparous, any combination of a risk-factor with previous PE or HBP reached a +LR>10. The combination of obesity and multiple pregnancy reached a good DA particularly in the presence of previous preeclampsia (positive likelihood ratio (LR+) = 26.5 or chronic hypertension (HBP) LR+ = 40.5. In primiparous, a LR+>15 was observed in multiple pregnancies with the simultaneous presence of obesity and diabetes mellitus or with HBP. Predicted probabilities above 97 centile in multiparous and 99 centile in primiparous provided high (LR+ = 12.5), and moderate (LR+ = 5.85), respectively. No one risk factor alone or in combination provided a LR- sufficiently low to rule-out the disease.In preeclampsia prediction the combination of specific risk factors provided a better discriminatory accuracy than traditional single risk approach. Our results contribute to a more personalized risk estimation of preeclampsia. This complication affects 3–10% of all pregnancies [Preeclampsia (PE) is defined as the new onset of hypertension and proteinuria after the 20gnancies –6. In faNICE guideline classifies women as at high-risk for PE when any major risk factor , autoimmune disease, diabetes mellitus (DM), or chronic hypertension (HBP)) or at least two moderate risk factors  are present[Task Force guidelines[World Health Organization guideline[For instance, the e present. The Tasuidelines, howeverguideline define hguideline.Overall, current screening guidelines are easy to apply in everyday practice as they are not based on complicated risk equations but mainly on the existence of one or two risk factors. The selection of those risk factors is mainly based on the difference in the average risk between exposed and non-exposed women, which is normally appraised by measures of association like the odds ratio (OR). However, this form of screening could be criticized, as previous methodological studies–12 have random forest analysis[A suitable approach to improve discriminatory accuracy in clinical decision making could be to adopt the perspective of precision medicine aiming t analysis and the This study is based on the Swedish Medical Birth Registry (MBR) that includes detailed and standardized information on nearly all pregnancies in Sweden culminating in delivery. Overall, the quality of the register is rather high as described elsewhere,16. Usinst January 2002 to 31th December 2010. We decided to restrict the analysis to deliveries from Swedish mothers that has been residing in Sweden for more or equal to 5 years in order to improve the homogeneity, completeness and validity of the information. To prevent that the same episode of PE counted twice in multiple pregnancies, only one children were randomly selected among those belonging to the same pregnancy. The flow diagram of the study population is shown in We identified all the 938.932 deliveries recorded in the MBR from 1th version (ICD-10). In this manuscript both pathologies are named PE. PE was recoded as positive whether it was present in the MBR or in the Patient register from 20 weeks of gestational age and up to six weeks after delivery. Gestational hypertension (ICD-10 code O13), chronic hypertension with proteinuria (O11) and edema and proteinuria (O12) were included as non-PE. Non-specified hypertensive disorder (O16) was considered as HBP. When the same patient was categorized as Gestational hypertension and PE was included as PE as previous studies with the MBR[The main outcome of our study was PE or eclampsia defined as a diagnosis coded O14 or O15, respectively, according the International Classification of Diseases 10h the MBR.2[All the exposure variables were dichotomized in order to follow a similar approach as in current clinical guidelines and categorized as: maternal age into <40 or ≥40 years, educational achievement as ≤11 years or ≥12 years of formal education obtained the year before the delivery and family situation as cohabiting with the child’s father or not. The presence of disease (ICD-10 codes) was identified at the first antenatal visit, at the hospital discharge after delivery and/or during a five-year period before pregnancy, and was considered as positive whether it was present in the MBR or in the Patient register. The codes obtained were: HBP (I10-I15)), DM ), CKD N18, N19)), and autoimmune diseases including systemic lupus erythematosus (M32) and rheumatoid arthritis . There were only 26 pregnancies with antiphospholipid syndrome (D686), therefore it was included together with autoimmune diseases. Obesity was defined as body mass index (BMI) ≥ 30kg/m2–19. Info), and auIn the MBR, the data concerning the current pregnancy is collected prospectively, before onset of potential adverse pregnancy outcomes, which prevents recall bias. PE diagnose is noted by the responsible doctor at discharge from hospital. The information about chronic hypertension and other maternal demographic, clinical and reproductive characteristics are recorded by a midwife during mother´s first visit for antenatal care. Then, the data is forwarded to the MBR where the information is computerized. The records are standardized and are identical throughout the country, which minimizes information bias.To decide whether to perform the analysis in the overall population or stratified by parity, we explored the interaction effect between each factor and parity by adding one interaction term to the baseline logistic model. Each model was specified as i = 1 if nulliparous and 0 if multiparous, and j = 1 when the risk factor was present and 0 otherwise. Then, OR11, OR10 and OR01 was obtained, OR00 was considered the reference category. Multiplicative interaction was determined by the ratio of Odds ratio (ratio of ORs = OR11 / (OR10 x OR01), directly obtained from the output of multiple logistic models. Additive interaction was determined by the relative excess of risk due to interaction relative to the risk without exposure also called Interaction contrast ratio (ICR = OR11−OR10−OR01+1), using the regression coefficients and covariance matrix obtained from the multiple logistic regressions. When the confidence interval did not include the value of one or zero, the interaction was considered statistically significant in the multiplicative or additive scale, respectively. The interaction was classified as positive in the multiplicative/additive scale if ratio of ORs>1/ ICR>0, negative if ratio if ORs<1/ICR<0 or absent if ratio of ORs = 1/ICR = 0. As significant modification was found in both multiplicative and the additive scales, all analyses were stratified on parity, also considering that previous PE can only be present in multiparous woman.The prevalence of risk factors in women with and without PE was calculated using point estimations and confidence intervals. Logistic regression analysis was performed to obtain crude and adjusted ORs stratified by parity. Starting from a saturated model containing all the variables, a backward elimination strategy was applied to construct the final multiple regression model. A significance level of 0.1 was defined to exclude variables from the saturated model. When the confidence interval did not contain the null, the difference was considered statistically significant. Since the data was correlated (children from different pregnancies clustered within mothers) we obtained robust standard errors and 95%CI. Models fit were compared by Akaike information criteria (AIC). Thereafter, a variable based on the number of current risk factors–6  single risk factors, 2) recreating three guidelines approach described above–7, 3) suThe absolute risk (AR), attributable risk (AF), true positive fraction (TPF), false positive fraction (FPF), and likelihood ratio (LR) were calculated. Briefly, the LR shows the probability of having or not the risk factor  in patients with PE and compare to the probability of the same results in patients without PE. The null value is 1. In general, a LR+ >10 is considered high enough to rule-in PE, 5–10 moderate and 2–5 small,22. The Preeclampsia was present in 3.83% of the population. The characteristics of the deliveries in multiparous and primiparous are presented in An interaction effect was observed between each traditional risk factors and parity, in one or both scales. The effect of risk factors on PE was lower in nulliparous than in multiparous. The lowest ICR ICR<2) was observed for the interaction of parity with HBP and with multiple pregnancy. An opposite direction of the interaction measures was observed between parity and diabetes and between parity and smoking.. 35.45%  was obseConcerning a single factor, a LR+ around 10 was only present in multiparous women with HBP or previous PE. No one of the studied variables showed a LR- lower than 0.2 Tables . Table 4Regarding primiparous, the combination of multiple pregnancy and obesity increased the risk particularly in the presence of DM. The combination of HBP and multiple pregnancy also presented a LR>10. A moderate LR was observed in those with HBP and obesity, DM and obesity and DM with multiple pregnancy. There were no patients with these 4 factors neither with the combination of DM, HBP and multiple pregnancy. In the absence of HBP, the combination of multiple pregnancy and obesity with gestational diabetes reached a moderate LR+  but a predictive value of 33%. In primiparous with no major risk factors, no one combination reached a LR>10 but a moderate LR was observed when multiple pregnancy was present in mother older than 40 or with obesity.The AUC and sensitivity for the model including all variables was higher in multiparous when compared to primiparous. Based omachine learning techniques  and stratification for parity, we inform the existence of individual heterogeneity in PE and identify specific combinations of specific risk factors with a high LR+ that permit a more assertive PE risk assessment for specialist referral and LDA prescription. Additionally, we demonstrate that a more extreme cut-off of the individual probabilities predicted by multiple logistic regression analysis is needed in primiparous when compared to multiparous in order to predict PE with confidence.Our population-based study confirms previous associations between PE and traditional risk factors, alone or in simple combinations. As recently recommended, we compOur results are compatible with previous findings reporting a positive association between PE and low education, maternaIn everyday clinical practice the definition of high risk women varies across guidelines. In some of them,6 the saApplying different approaches  we identified specific combination of risk-factors that provided a higher discriminatory accuracy to rule-in PE with confidence. For instance, among multiparous, any bivariate combination including HBP or previous PE reached a LR>10. There are few studies analyzing the impact of combinations rather than a single factor in PE risk. Interesting, despite CKD has been considered a major risk factor for PE, our results are in accordance with a previous study reporting that only those with the simultaneous presence of HBP are at a higher risk of PE. We addiIn primiparous, only “rare” combinations of risk factors achieve a LR>10, particularly multiple pregnancy with HBP or with the simultaneous presence of DM and obesity. However, some combinations with a moderate LR provided an absolute risk above 30%, i.e. obesity with HBP or with DM. As observed in multiparous, the combination of multiple pregnancy and obesity was associated with an increased OR and LR+, particularly among those without major risk factors. This finding might suspect the role of volume overload in the pathogenesis of PE as a hypertensive disease,35. OtheThe contribution of traditional risk-factors to the DA for PE was modest as previously reported. Using dDespite the improvement in model fit when the subgroups were incorporated in multiple regression models, the sensitivity (TPF) and specificity (TNF) remained constant. This can be explained by the low prevalence of high-risk subgroups in the population. Contrarily to general belief, the TPF and the FPF depend of the prevalence ratio. That isSome researchers have pointed out that measures of association alone are unsuitable for discriminatory purpose,23,42. OTo our knowledge, this is the first study explicitly focused on understanding heterogeneity in PE risk in order to identify high-risk subgroups of PE by combining specific risk-factors. While most previous studies have provided risk equations for the whole population of women,44 or maThis study also has potential limitations. First, we have excluded missing data, but we cannot assure that missing values were completely at random even if included and excluded deliveries were balanced concerning the prevalence important risk-factors. Second, we had no information on interventions during pregnancy, such as aspirin prophylaxis. At the study interval, there was no preeclampsia screening in place, however, some patients at risk were possibly on LDA treatment, particularly women with prior preterm preeclampsia, i.e., probably around 2%-5% of all preeclampsia cases, which might bias the estimations towards the null in this population. Third, we were not able to evaluate all possible combinations of moderate risk-factors reported in published guidelines, neither include the histories of previous PE from longer pregnancy intervals. Fourth, we have not validated our finding in a separate population or by mean of bootstraps so we cannot rule out that our multiple regression model might be overfitted and the AUCs overestimated. This is particularly important with the results from the smaller subgroups that may be the product of overfitting and may not readily reproduce in other study samples. We have used dichotomous variables in other to adopt a similar approach as that used in current guidelines, however ordinal or continuous variables could produce more precise estimations. Finally, even though the quality of the MBR seems appropriate, our resNo one risk-factors alone or unspecific combinations reached an acceptable accuracy, and ≥3 moderate risk combinations are needed in those without major risk-factors to reach a LR+> 10. Consequently, current approach based exclusively in OR, might be associated with inefficient specialist referral and unnecessary treatment with LDA. The prediction of PE was improved with a more individualized approach, by identifying specific combinations or by defining a differential cut-off to the distribution of the predicted probability for multiparous and nulliparous obtained by multiple regression analysis. However, the absence of any single neither relevant combinations were enough to rule-out the disease. The identification of such specific subgroups can improve the reliability of LDA prescription, but those with any single risk might need further screening. Our results contribute to a more personalized risk estimation of preeclampsia.S1 Table(DOCX)Click here for additional data file.S2 TableOne interaction term was included in each model. Previous preeclampsia was considered a dummy variable with three categories: yes or no in multiparous and no applicable in primiparous.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.