Industrial companies indicate a tendency to eliminate variations in operator strategies, particularly following implementation of the lean principle. Companies believe when the operators perform the same prescribed tasks, they have to execute them in the same manner (completing the same gestures and being exposed to the same risk factors). They attempt to achieve better product quality by standardizing and reducing operational leeway. However, operators adjust and modify ways of performing tasks to balance between their abilities and the requirements of the job. This study aims to investigate the variability of exposure to physical risk factors within and between operators when executing the same prescribed tasks. The Ergonomic Standard method was used to evaluate two workstations. Seven operators were observed thirty times between repeated cycle times at those workstations. The results revealed the variability of exposure to risk factors between and within operators in the repeated execution of the same tasks. Individual characteristics and operators’ strategies might generate the variability of exposure to risk factors that may be an opportunity to reduce the risks of work-related musculoskeletal disorders (WR-MSDs). However, sometimes operators’ strategies may cause overexposure to risk factors; operators most often adopt such strategies to undertake their tasks while reducing the workload. Prevention of work-related musculoskeletal disorders (WR-MSDs) remains a challenge in the industrial settings. Establishing a successful prevention approach consisting of different workplace interventions might reduce the onset or prevalence of WR-MSDs ,2. OrganPhysical variation can be separated into different types . ExtrinsIndustrial companies show a tendency to eliminate operational leeway, particularly following implementation of the lean principle. A trend in automotive industries indicates the increase of work standardization (use of element sheets for workstations), best practice (performing the tasks in the same way), and limiting operational leeway (coping strategy) . FurtherThe challenge is whether manufacturers should take into account the variability of exposure to physical risk factors due to operational leeway in design and production. By limiting operational leeway, they believe that operators have to perform their tasks in the same manner, and the current assessment approach overlooks the variability of exposure by assessing only one operator in specific cycle time. This study, therefore, aims to investigate the variability of exposure to physical risk factors within and between operators in repeated executions of the same prescribed tasks. Following ergonomic research performed over three years in one sector (including eleven assembly workstations) of a truck assembly plant in France , we seleRepetitionPosture including work posture, access-hidden assembly, clearance for hand-finger, workspace for hand, handgrip, surface area for pressure, component size, static back posture, static neck posture, static shoulder posture, wrist postureMaterial-handling including two-handed lifts and one-handed liftsForce including pushing/pulling (whole body force), pushing/pulling with the hand/arm, pushing/pulling with fingerEnergy consumption including movement (continuous steps), climbing/stepping over, and tightening torque.The in-house Ergonomic Standard method  was usedThe observational tool prioritizes the identified risk factors, qualitatively based on a traffic light model-green: Minimum risk, yellow: Moderate risk, and red: Excessive risk . Each suOperator one and two were exposed to a higher number of red assessments (6 reds) at the “Preparation of bumper workstation” compared to the experienced operators (4 reds) evaluated in our previous study . The expOperator one exposed to more risk factors on Monday and Wednesday than on Tuesday at the mounting SCR workstation, but operator two had more exposure on Tuesday at this workstation. Operator two at the mounting SCR workstation showed a higher variability of exposure than the other operators. “Access, hidden assembly” risk factors differed for operator one, two and three between the executive CTs on the same day. For example, this risk factor for operator two varied from green to red in four CTs on Tuesday, but it was yellow in four CTs assessed on Friday. We observed the similar results for the “pushing/pulling with hand, arm” risk factor for this operator, but the variation was on Friday. The assessment of this risk factor was the same  for operator one on the same day. However, its assessment for operator three and four was different in two CTs evaluated on the same day . We found a variability of exposure to physical risk factors between and within operators in the repeated execution of the same prescribed tasks. Our findings show a higher variability of exposure to the number of red and yellow assessments between and within operators at the Mounting SCR Tank workstation. The characteristics of the workstations might be a reason for the difference in the variability of exposure. The more red and yellow assessments were found in a workstation, the more operators used different strategies for performing the tasks, which led to more variability of exposure. Gaudez et al. (2016) in a review article mentioned that work characteristics are the source of variability .Exposure to physical risk factors at both workstations was higher in this study than our previous study in which we evaluated only an operator in one cycle time . It mighWe evaluated all of the participants at the same period of the day (10–12 a.m.) but not during the same day of a week. It was impossible to evaluate all of the cases on the same day because we needed the operator to work on the frequent type of truck in a given workstation between 10 a.m. and 12 a.m. These conditions were often impossible in a real setting. These results could not confirm that physical risk factors decrease or increase with the execution of consecutive cycle times across the different days.The operators in this study executed their tasks differently, which might relate to operational leeway in the workstations. Compared to the typical automotive industries, these workstations provided more operational leeway because of various tasks in a cycle and more cycle time. A coping strategy due to having operational leeway for performing a job might be a reason for the variability of exposure between operators, as this variability was high in posture category risk factors of our assessment tool. Recent studies have shown that coping strategies enable operators to adapt and regulate their gestures and movements, which might be beneficial for reducing work-related musculoskeletal pains ,14,15,16The variability of exposure found in this study might also associate with motor control variability (intrinsic variability) ,11,12,20Our results show that the current approach of WR-MSD risk measurement based on the assessment of a workstation and an experienced operator is a debatable one. Assessing different operators in several cycle times proved that the type and level of exposure changed with the primary assessment performed in our previous study . The praA possible limitation of this study is that we assessed the operators during different days and the variability of exposure might be related to the mood of an operator during that specific time instead of being related to their coping strategy and motor variability. For example, operator two at the Mounting SCR tank workstation had several red risk factors on Tuesday, while his assessment had less red risk factors on Friday. The psychological conditions of the operator may influence on his activities in different days. Furthermore, we could not include the same sample of observations for the participants, but we attempt to have at least two observations on the same day for a participant. The difference in the number of observations between both workstations might influence the variations of yellow and red assessments.This study shows the variability of exposure to physical risk factors between and within operators in the execution of the same prescribed tasks. Our results confirm that the current approach for assessing WR-MSDs risks may misestimate risk levels because the assessment is for one specific operator during a given cycle time. These findings justify the idea that manufacturers should consider inter and intra-individual variability of exposure to physical risk factors in the design and production. This study could not answer the question of whether the variability of exposure relates to coping strategy or motor control variability. Further investigation is needed to study the relationship between the variability of exposure and motor control variability. Additional research is essential, particularly in automotive assembly plants, to consider the variability of exposure and operational leeway in the WR-MSDs risk assessment phase.


Sitting posture monitoring systems (SPMSs) help assess the posture of a seated person in real-time and improve sitting posture. To date, SPMS studies reported have required many sensors mounted on the backrest plate and seat plate of a chair. The present study, therefore, developed a system that measures a total of six sitting postures including the posture that applied a load to the backrest plate, with four load cells mounted only on the seat plate. Various machine learning algorithms were applied to the body weight ratio measured by the developed SPMS to identify the method that most accurately classified the actual sitting posture of the seated person. After classifying the sitting postures using several classifiers, average and maximum classification rates of 97.20% and 97.94%, respectively, were obtained from nine subjects with a support vector machine using the radial basis function kernel; the results obtained by this classifier showed a statistically significant difference from the results of multiple classifications using other classifiers. The proposed SPMS was able to classify six sitting postures including the posture with loading on the backrest and showed the possibility of classifying the sitting posture even though the number of sensors is reduced. Ergonomic information provided for the seated person plays a crucial role in improving the sitting posture by changing the habits and attitude of the seated person ,2,3,4,5.A recent combination of IT technology and various sensors has enabled a sitting posture monitoring system (SPMS) to assess the posture of the seated person in real-time and to improve sitting posture. Previous studies on SPMSs can be classified according to two purposes. The first purpose is to determine whether or not a person is seated on the office chair, which is used mainly to monitor seat occupancy ,7,8,9. TRecent advances in machine learning have led to the use of machine learning algorithms in many studies. Machine learning algorithms have the advantage of minimizing errors by training themselves through optimization and tuning and have recently been used in various areas, such as fall detection , classifSeveral studies on sitting posture estimation have proposed SPMSs based on various sensors and have rarely been commercialized because of the high cost of the measuring devices. The prices of products could increase because of the measurement of all loads applied to the sensors that were attached to both the backrest plate and seat plate , or by aPrior to the experiment, the representative sitting posture was defined with reference to previous studies on sitting postures. From these previous studies, the present study derived representative sitting postures, such as the sitting posture in the forward position, middle position, or backward position, forward sitting posture, reclined sitting posture, slumped sitting posture, laterally tilted left or right sitting posture, and crossed legs right over left or left over right sitting posture ,9,16,17.In this study, an SPMS was fabricated by replacing the seat frame of an existing office chair with a new frame with the load cell inserted . The seaThis study involved 24 healthy adult males . The selected subjects regularly worked with a video display terminal, sitting on office chairs for eight or more hours a day, and had no apparent severe musculoskeletal deformity or nervous system abnormality. This study was conducted with the approval of the Bioethics Committee of the Public Health Agency designated by the Ministry of Health and Welfare, South Korea (IRB P01-201607-11-001).The experiment was divided into a preliminary test (15 subjects) and the main test (nine subjects). The 15 subjects were pre-tested to select an SPMS estimation and definition of the BWR range for the sitting posture and in a preliminary test. The other nine subjects participated in the main test for classification using machine learning. For the preliminary test, the load values measured from the load cells of the SPMS were obtained according to the six sitting postures, and the BWR algorithm was defined. Before the experiment, the height of the chair was adjusted to the popliteal height of the subjects, who were fully trained on the classification of the six postures. The subject sat on the office chair according to the instruction on the sitting posture. When the posture was compliant with the instruction, the subject held this posture for 30 s with his arms crossed, and then stood up from the chair. Each posture was repeated five times. For the main test, a total of six sitting postures were randomly changed. The sitting posture was changed every 10 s, and the information regarding each sitting posture was presented with texts and images as well as an auditory signal. After the subject changed the posture according to an auditory signal, the subject maintained the posture with his arms crossed while repeating the procedure. A total of 30 posture changes during each trial were processed from the previous sitting posture to the next sitting posture and derived from the combination of the six sitting postures. The posture change process was designed to avoid an overlap between the previous and succeeding postures. Thus, each trial comprised a total of 30 posture changes over 5 min, and there were a total of three trials (90 posture changes over 15 min). All three trials took place continuously for each of the subjects.SUMR, Equation (1)), a ratio of distribution in the medial-lateral direction ), and a ratio of distribution by the anterior-posterior direction ). The BWR area determined through the preliminary test is shown in MLR was used to measure lateral leaning, which was divided into LE, RI, and the other four posture groups  (SUMR and APR resulted in four different posture groups (BW represents the body weight.The weight (kg) data measured by the four load cells of the SPMS were transferred to a PC at a rate of 1 Hz via the Arduino board. For the preliminary test, the load data collected from the four load cells were classified into a ratio of SPMS to body weight  a. Furthee groups b accordix, Support vector machines (SVMs)  are the The distance between the hyperplane and support vector can be calculated as follows:Thus, the margin, the distance between the two support vectors, is represented as follows:K is defined as follows:According to Equation (7) above, the margin becomes maximum when ear data . SVMs wiUsing Equation (9), Equation (4) above can be modified as follows:The kernel function used in this study can be expressed as a radial basis function (RBF) as follows:To verify whether an SVM using the RBF kernel functions best in the system proposed in this study, an SVM using a linear kernel, linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), Naïve Bayes, and a random forest classifier were applied to the data. The LDA is a classification algorithm to find a hyperplane that classifies dependencies by minimizing within-class scatter and maximizing between-class scatter ,21. The A test using the random forest classifier was conducted to determine the number of trees when the classification rate reaches the saturation point. Consequently, we could ascertain the optimal performance with the minimum number of trees. c binary classifiers that code the i class to 1 and all other classes to −1. In the c-class classification problem, the method learns several binary classifiers for multiple classifications by selecting one class and setting all the remaining classes as a different class [Since the SVM is a binary classifier, the one-against-all strategy was used to extend the SVM to a multi-classifier. The one-against-all strategy is a method used to create nt class . The mult-test was performed to calculate a p-value to determine whether there was a statistically significant difference between the results from the SVM using the RBF kernel and those of the other classifiers, and a significant difference was noted (p < 0.001).All classifiers were trained using the training data, which contain the six sitting posture changes. The classification accuracy was calculated using the test data, which contain the remaining 84 sitting posture changes. To determine if the number of sensors used could be further reduced, the sitting position was classified using different classifiers after excluding a specific sensor among the four load cells used. In this study, we proposed a system for monitoring six sitting positions by mounting only four low-cost load cells onto the seat plate of the chair, unlike several studies on the SPMS reported to date, which used several load cells mounted on the seat plate and backrest of a chair. As shown in p < 0.05) compared to the results from the other classifiers evaluated.The study used seven classifiers  to classify the sitting postures via the load cells. The results suggest that the SVM using the RBF kernel is a suitable method for classifying sitting postures with sensors exclusively on the seat plate; the kernel had an average classification rate of 97.20%. Furthermore, the results from the SVM using the RBF kernel showed a statistically significant difference , which can improve the computation time and power consumption of the hardware in order to classify sitting postures in real-world settings. Moreover, in this study, environmental variables were limited in order to maximize the accuracy of the system. Future studies shall explore variations in posture . Finally, as shown in


Electrocardiogram (ECG) signals contain a great deal of essential information which can be utilized by physicians for the diagnosis of heart diseases. Unfortunately, ECG signals are inevitably corrupted by noise which will severely affect the accuracy of cardiovascular disease diagnosis. Existing ECG signal denoising methods based on wavelet shrinkage, empirical mode decomposition and nonlocal means (NLM) cannot provide sufficient noise reduction or well-detailed preservation, especially with high noise corruption. To address this problem, we have proposed a hybrid ECG signal denoising scheme by combining extreme-point symmetric mode decomposition (ESMD) with NLM. In the proposed method, the noisy ECG signals will first be decomposed into several intrinsic mode functions (IMFs) and adaptive global mean using ESMD. Then, the first several IMFs will be filtered by the NLM method according to the frequency of IMFs while the QRS complex detected from these IMFs as the dominant feature of the ECG signal and the remaining IMFs will be left unprocessed. The denoised IMFs and unprocessed IMFs are combined to produce the final denoised ECG signals. Experiments on both simulated ECG signals and real ECG signals from the MIT-BIH database demonstrate that the proposed method can suppress noise in ECG signals effectively while preserving the details very well, and it outperforms several state-of-the-art ECG signal denoising methods in terms of signal-to-noise ratio (SNR), root mean squared error (RMSE), percent root mean square difference (PRD) and mean opinion score (MOS) error index. An electrocardiogram (ECG), derived from the body’s surface, indicates the rhythmic electrical activity of myocardia. The interpretation of abundant physiological and pathological information on the heart contained in ECG recordings provides a noninvasive technique for cardiovascular disease diagnosis. However, ECG signals are usually contaminated with baseline wander, high-frequency noise caused by electromyography, and motion artifacts during collection and transmission. Since these contaminants overlap the cardiac component in both the spatiotemporal and frequency domains, the extraction of weak cardiac components from the corrupted ECG signals and the preservation of the subtle features of the signals are fairly difficult. Indeed, ECG signal denoising is very important for improving the quality of ECG signals, thereby facilitating subsequent signal processing and analysis tasks such as ECG watermarking, compression , featureNumerous ECG signal denoising methods have been proposed. These methods can be generally divided into three categories, i.e., spatiotemporal domain methods, frequency domain methods and statistical techniques. Classical low-pass filters ,5, mediaDenoising methods based on empirical mode decomposition (EMD) , ensemblStatistical techniques such as independent component analysis , principIn 2005, Buades et al.  proposedTo address these issues, we have proposed to combine the extreme-point symmetric mode decomposition (ESMD)  with theM is the number of IMFs and R is the final residue which will be optimized to be an optimal AGM curve.The ESMD has been recently proposed by Wang et al. as a modification of the EMD. The ESMD decomposes the signal into a series of intrinsic mode functions (IMFs) and a final residue which is an optimal adaptive global mean (AGM) curve. The IMF is the basic oscillation mode and the AGM curve reflects the evolutionary trend information of the signal. Different from the EMD, the ESMD allows the existence of a certain number of extreme points in the final residue and it determines the best sifting times to obtain the IMFs by optimizing the AGM curve. Using the ESMD method, the ECG signal The detailed implementation of the ESMD based ECG signal decomposition involves the following steps.Step 1: Find all local extrema points of the ECG signal Step 2: An interpolating curve based on all the midpoints T or 1.Step 3: Steps 1 and 2 should be repeated with Step 4: Repeat above steps with T in a finite integer interval from Tmin to Tmax. Choose T0 with the smallest variance ratio Step 5: Repeat the above steps with different T0. In this way, we will obtain all the IMFs and the final residue R.Step 6: Repeat step 1 to step 4 with the optimal sifting times 1 contains merely high frequency noise component and there is no apparent correlation between IMF1 and the ECG signals. IMF2 consists of QRS complex together with high-amplitude noise. IMF3~IMF5 are slightly stained by high-frequency noise. Other IMFs and AGM contain the low frequency components, and they involve almost no noise. The decomposed results of the ECG signal indicate that it is desirable to only denoise IMF1~IMFi while leave IMFi+1~IMFn and the residual R unprocessed to ensure effective noise reduction and good detail preservation.To demonstrate the effect of the ESMD method in decomposing the ECG signal, 1 and IMF2 as shown in 1 and IMF2, it is preferable to extract the exiguous QRS complex components while suppress the noise. However, this is a very difficult task. If the NLM filter is directly implemented on them, the noise and the ECG components will be removed simultaneously. Therefore, we propose to detect the location of QRS complexes by the wavelet based detection algorithm. Using this algorithm, we will obtain the set P of points in the QRS complexes.When the ECG signals are contaminated by high levels of noise, the noise will be decomposed into IMF1 and IMF2, and the singularity is found in the wavelet domain. Then the mean amplitudes of the poles are obtained by setting the threshold of R wave to be 30% of the difference value between the maximum amplitudes and the minimum ones. Finally, the distance between two adjacent maxima is computed. If this distance is less than 0.4, the maxima with lower amplitude will be removed. In this way, the points in the R wave will be obtained.As regards the detection of R wave, the wavelet transform is applied to IMFFor the detection of the Q wave, if the wave is a forward/inverted wave, a minimum/maximum forward from the R wave is found in the wavelet transform domain. The detection of the S wave is similar to that of the Q wave. The only difference is that the S wave is found backward from the R wave in the wavelet transform domain.For ECG signal denoising, the NLM method restores a given point using the weighted average of point intensities within a search window Ω, where the weights are determined based on the similarity between two windows centered at the two considered points. The principle of the NLM method is shown in ith point by the NLM method is computed as:Let the ECG signal The decay parameter has an important influence on the restoration performance of the NLM method. To expound the choice of this decay parameter for the effective ECG signal denoising, we will introduce the bias-variance principle . Based oj. Inspired by the work in [j as L in the noisy signal The two items rding to , bias2 method, we have chosen four other denoising methods for comparison including the NLM method , the VMDor index . The higOS tests . The bli signals . The othAs regards the NLM method, the decay parameter is fixed at 1.1http://oset.ir/category.php?dir=Tools. For the simulated ECG signals, Gaussian white noise with the variance To demonstrate the advantage of our method, we will focus the performance evaluation on the restored results of smooth segments and the ECG’s amplitudes. From error values for all evaluated methods. The mean MOSerror is computed as the mean of all MOSerror values for each compared denoising method when error values. Here, it should be noted that the MOSerror value of the MED method reaches 30 while the MOSerror values of the ESMD-NLM method and the VMD and EEMD methods are lower than 15 and 30, respectively. Based on the classification criterion of the signal quality in [To further demonstrate the effectiveness and advantage of the proposed method in removing other kinds of noise, we have conducted experiments on the first group of simulated ECG signals corrupted by muscle artifact noise with the variance of 60. Here we will only make comparisons among the ESMD-NLM method, the VMD method, the EEMD method and the NLM method because the latter are closely related to our method. error comparisons for the ESMD-NLM method and the other three compared methods operating on the first group of simulated ECG signals corrupted with MA noise. As shown in error values are higher than those of the proposed method. Please note that the MOSerror values of the VMD and EEMD methods are higher than 50, which indicates the very bad quality of the denoised signals for the two methods. Likewise, the NLM method provides the bad quality denoised signals because its MOSerror value reaches 30. By comparison, the proposed method can provide the denoised signals with good quality. Indeed, the data in this table illustrate that the ESMD-NLM method can suppress muscle artifact noise and preserve the details better than other methods. To demonstrate the practicality of the proposed method, we have applied it to denoising the three groups of real ECG signals. Here, we have selected the 100m.dat, 111m.dat and 221m.dat from MIT-BIH Arrhythmia Database as the first group, the sel100m.dat, sel117m.dat and sel114m.dat from QT database as the second group, and the 13420_12m.dat, 13649_04m.dat and 12713_04m.dat from MIH-BIH ECG Compression Test Database as the third group. For the real ECG signals in units of mV, the gain is 200 and 400 for the first and second groups and the third group, respectively. The base is 1024 and the sampling frequency is 360 Hz for the first group while the base is 0 and the sampling frequency is 250 Hz for the second and third groups.To evaluate the performance of these compared methods operating on the real ECG signals, the MOS tests are also implemented. Because the original noise-free versions of these real ECG signals are unknown, only the clinical blind MOS tests are done by the three cardiologists without knowing the source of each tested signal. For the blind MOS tests, only the general quality score of the denoised signals is obtained for each cardiologist as described in  and the To compare the computational efficiency of all evaluated methods operating on the 100m.dat, sel100m.dat and 13420_12m.dat, In this paper, we have proposed a novel ECG signal denoising method by combining ESMD with NLM. In the proposed ESMD-NLM method, ECG signals are decomposed into many IMFs with different frequencies, and then the IMFs are denoised by nonlocal means using different decay parameters based on the QRS detection results. Quantitative comparisons based on the simulated ECG signals demonstrate that the ESMD-NLM method performs better than the median filter, the wavelet based method, the EEMD method and the NLM method by providing lower PRD, RMSE and MOS error values as well as higher SNR values. Visual comparisons based on the simulated and clinical ECG signals indicate that the proposed method gains an advantage over the compared methods in terms of noise suppression and detail preservation. Future work will be focused on the extension of the ESMD-NLM method to denoising other physiological signals such as electrooculogram (EOG), electroencephalograph (EEG) and electromyogram (EMG) signals.


Continuous cardiac monitoring has been developed to evaluate cardiac activity outside of clinical environments due to the advancement of novel instruments. Seismocardiography (SCG) is one of the vital components that could develop such a monitoring system. Although SCG has been presented with a lower accuracy, this novel cardiac indicator has been steadily proposed over traditional methods such as electrocardiography (ECG). Thus, it is necessary to develop an enhanced method by combining the significant cardiac indicators. In this study, the six-axis signals of accelerometer and gyroscope were measured and integrated by the L2 normalization and multi-dimensional kineticardiography (MKCG) approaches, respectively. The waveforms of accelerometer and gyroscope were standardized and combined via ensemble averaging, and the heart rate was calculated from the dominant frequency. Thirty participants  were asked to stand or sit in relaxed and aroused conditions. Their SCG was measured during the task. As a result, proposed method showed higher accuracy than traditional SCG methods in all measurement conditions. The three main contributions are as follows: (1) the ensemble averaging enhanced heart rate estimation with the benefits of the six-axis signals; (2) the proposed method was compared with the previous SCG method that employs fewer-axis; and (3) the method was tested in various measurement conditions for a more practical application. Continuous cardiac monitoring is necessary for the diagnosis of cardiovascular disease. A medical emergency caused by a cardiac arrest can be prevented by continuous cardiac monitoring especially from wearable devices. In recent years, the advancement of novel instruments has allowed the monitoring of cardiovascular activity outside of clinical environments. One of the vital components to develop the monitoring system is seismocardiography (SCG). SCG is non-invasively measured from the thoracic movements produced by the contraction of heart and the ejection of the blood from ventricles into the vasculature  using anInitial studies on SCG only focused on the z-axis accelerometer signal ,2. They Recent SCG studies have investigated the gyroscope signal to enhance the cardiac monitoring. Tadi et al.  proposedDespite the steadily proposed methods of the novel cardiac indicators, SCG has presented lower accuracy compared to traditional methods . It is necessary to develop an enhanced method by combining the significant cardiac indicators. The statistical methods have been applied to reduce noise and amplify deterministic signals from random signals. The ensemble averaging for repeatedly measured accelerometer signals have been demonstrated to reduce noise on SCG , which aConsequently, recent studies on SCG have been aimed for more distinct measurements by combining the additional information of motion relevant to heartbeat. However, they tried to combine multi-axis of the same sensor not other sensors, even though the peaks of the acceleration and angular velocity of thoracic movements had associated each other. Thus, this study hypothesized that the accuracy of SCG can be increased by combining the accelerometer and gyroscope signals, which is the significant cardiac indicators represented acceleration and angular velocity of the thoracic movements derived from heartbeat.This study was conducted to develop a novel method to combine the six-axis accelerometer and gyroscope signals and to enhance practical application of the cardiac monitoring. The focus was on the heart rate estimation to demonstrate the proposed method, since it is the most popular indicator to monitor the cardiac activity. The signals are sensitive to physical conditions and postures. Therefore, they were experimented to estimate the heart rate in the relaxed and aroused conditions during standing and sitting postures. The ECG was simultaneously measured to verify the accuracy of heart rate estimation from SCG. The proposed method combined the six-axis accelerometer and gyroscope signals. It was compared with the traditional SCG method, which only employed the z-axis or tri-axis accelerometer signal. The contributions of this study can be summarized as follows: (1) the ensemble averaging enhanced heart rate estimation with the benefits of the six-axis signals; (2) the proposed method was compared with the previous SCG method that employs fewer-axis; and (3) the method was tested in various measurement conditions for a more practical application.A power analysis was conducted to determine the required number of samples for valid inference in this study. Two-independent Pearson’s correlation was used to assess the hypothesis. The power analysis was conducted using G * Power . In ordex-axis is the left-to-right direction, y-axis is the foot-to-head direction, and z-axis is the dorso-to-ventral direction.This study measured SCG from the chest utilizing a portable and wearable sensor, which has the MEMS accelerometer and gyroscope. The apparatus was comprised of three modules such as an Intel Edison, a Sparkfun 9DOF, and a Sparkfun Battery see . Intel EOne of the challenges for SCG is that the accelerometer signal is sensitive to body postures. To ensure clinical relevance, it is necessary to test SCG for a wide range of heart rates. Therefore, this study experimented the proposed method in various physical conditions and postures, as shown in SCG was measured when participants were physically relaxed or aroused during standing or sitting postures. To ensure relaxation, participants were asked to close their eyes and maintain body posture for 3 min. For arousal, participants were asked to maintain body postures for 3 min after running at a speed of 8.5 km/h on a treadmill for 3 min. These procedures were repeated twice during two different body postures . The experimental procedure was 30 min long and sufficient rest was given between each tasks. Longer rest time was given after arousal tasks due to the time for cardiac activity to be restored back to steady condition. This protocol was approved by the Institutional Review Board of the Sangmyung University, Seoul, Korea (BE2016-14).The accelerometer and gyroscope signals were measured with the SCG measurement device developed in this study, and the ECG was measured by the Lead-I simultaneously. The ECG served as a ground-truth for the evaluation of SCG. The ECG was measured at sampling rate of 512 Hz by a system consisting of a ECG 100C amplifier system and a MP150 data acquisition system . Participants were asked to minimize the movement of their arms to reduce the noise while measuring the ECG.The accelerometer and gyroscope signals were recorded to a local flash storage in our apparatus for minimizing data loss, even though they can be measured by connecting to another Bluetooth device in real time. In addition, the system for data acquisition has been operated with the multi-threading for real-time data processing in the future. After the experiment, the signals were analyzed on a desktop with Python. SCG was determined by analyzing and combining both the accelerometer and gyroscope signals as described below.First, the raw signals were measured with the sampling rates within the ranges of around 100 and 200 Hz. The sampling rates for the accelerometer and gyroscope are slightly different due to the data acquisition system based on multi-threading. Their sampling rates were fixed at 256 Hz to match the length of the signals for their integration. This study employed the cubic spline interpolation , which eSecond, the motion-induced noise was estimated by the Savitzky-Golay filter  . BecauseThe noise was subtracted from each signal to reduce the motion artifacts. The Savitzky-Golay filter is able to preserve higher order moments around inflection points , thus, iThird, the kinetic energy waveforms of accelerometer and gyroscope were calculated from tri-axis signals of accelerometer and gyroscope by L2-normalization and multi-dimensional kineticardiography (MKCG) approaches , respectposed by . Then, tposed by , and theForth, SCG was finally determined by the normalization and ensemble averaging of the energy waveforms of the accelerometer and gyroscope. The waveforms were standardized by the z-score normalization and combined via the ensemble averaging:In SCG studies, it was important to select appropriate peak detection algorithms according to the shape of the signals for heart rate estimation. Depending on the type of axis, the peak detection algorithms showed different performances . In thisOn the other hand, the ECG was clearly measured without noise because the participants were asked to minimize the movement of their arms in experiments. Thus, the heart rate of the ECG was calculated by the QRS detection algorithm, which was implemented by Pan and Tompkins to detect the R peaks . The QRSp-value was less than 0.05. The Bland-Altman plot was statistic method that was used for the comparison between values which were achieved by two measurement techniques. This method was consistent with the analysis, which combined both the graphical and statistical interpretation. The plot was represented by assigning the mean (x-axis) and difference (y-axis) between the two measurements. The 95% limits of an agreement were calculated by mean difference and the ±1.96 standard deviation of the difference was represented as lines on the plot.The heart rate with SCG was evaluated by comparing it to the heart rate with the ECG, which was based on their mean absolute error (MAE), standard deviation of absolute error (SDAE), and root mean squared error (RMSE), Pearson’s correlation coefficients (CC), and Bland-Altman plot. The MAE, SDAE, and RMSE were utilized as quantities for evaluating the similarity between predictions and observations. If the calculated error was approaching to the 0, it was indicated as the observations were similar with the predictions. The Pearson’s correlation coefficient was utilized to determine the statistical relationship between the two groups. The correlation coefficient approaching to the 1 was indicated as a strong positive correlation, whereas the value approaching to the −1 indicated as a strong negative correlation. The correlation was indicated as statistically significant if The heart rates measured from z-axis of accelerometer, tri-axis of accelerometer, and six-axis of accelerometer and gyroscope were evaluated with respected analyses. The Bland-Altman plots of estimated heart rate during standing and sitting postures in relaxed condition are shown in The heart rates during standing and sitting postures in aroused condition were evaluated as shown in The In this study, the heart rate was estimated from SCG of the z-axis, tri-axis, and six-axis in the relaxed and aroused conditions during standing and sitting postures. This study evaluated the accuracy of the proposed method, which combines the six-axis accelerometer and gyroscope signals, and compared with the traditional method which only employ the z-axis or tri-axis accelerometer signal. The performance of our method was much higher than the traditional method in all physical conditions and body postures.Overall, this research has drawn four significant findings. First, the heart rate from only the accelerometer was more accurately detected when analyzing multiple axes  than when analyzing the single axis . This result shows that the x- and y- axis of accelerometer also reflect the thoracic movement induced by heartbeat as much as z-axis of one. Although this result has been hypothesized in previous study , the perSecond, the heart rate in aroused condition was detected more accurately than in relaxed condition during both standing and sitting postures. This can be interpreted that the accelerometer and gyroscope signals could reflect strong heartbeat better than weak heartbeat due to the large movement of the chest. Thus, it is necessary to analyze the characteristics of the accelerometer and gyroscope signals depending on the physical conditions. If the signal processing was adaptively selected based on the characteristics, the estimation of heart rate may be enhanced. Recent studies by Migeotte et al. , CastiglThird, this study observed significant results for integration of the six-axis. Particularly, their correlation coefficients were higher than results of z-axis and tri-axis. This result indicated that with the advancement of the signal processing for the six-axis, the ECG could eventually be replaced by SCG. Future studies on SCG should consider the use of additional motion sensors, which include the gyroscope.Finally, this study presented the combination of the accelerometer and gyroscope to improve the accuracy of heart rate estimation from SCG. Despite the low accuracy of our results, a new apparatus was developed for the convenience of a more practical use of SCG in daily life. Previously, wearable SCG measurement systems have been tightly worn on the chest especially on the chest area, near the clavicle ,18,19, bThis study developed a novel method to estimate the heart rate from SCG by ensemble averaging of the six-axis of accelerometer and gyroscope. Proposed method estimated the heart rate more accurately than the traditional SCG methods in relaxed and aroused conditions during standing and sitting postures. Former SCG measurement methods have used accelerometer and gyroscopes, but they did not focus on combining the two to improve the estimation accuracy. This study implied that a reliable measurement method for integrating multi-signals should be established. The proposed method is expected to increase estimation accuracy of the heart rate, and therefore it takes further steps toward the development of SCG.


The entorhinal cortex (EC) is the major input and output structure of the hippocampal formation, forming the nodal point in cortico-hippocampal circuits. Different division schemes including two or many more subdivisions have been proposed, but here we will argue that subdividing EC into two components, the lateral EC (LEC) and medial EC (MEC) might suffice to describe the functional architecture of EC. This subdivision then leads to an anatomical interpretation of the different phenotypes of LEC and MEC. First, we will briefly summarize the cytoarchitectonic differences and differences in hippocampal projection patterns on which the subdivision between LEC and MEC traditionally is based and provide a short comparative perspective. Second, we focus on main differences in cortical connectivity, leading to the conclusion that the apparent differences may well correlate with the functional differences. Cortical connectivity of MEC is features interactions with areas such as the presubiculum, parasubiculum, retrosplenial cortex (RSC) and postrhinal cortex, all areas that are considered to belong to the “spatial processing domain” of the cortex. In contrast, LEC is strongly connected with olfactory areas, insular, medial- and orbitofrontal areas and perirhinal cortex. These areas are likely more involved in processing of object information, attention and motivation. Third, we will compare the intrinsic networks involving principal- and inter-neurons in LEC and MEC. Together, these observations suggest that the different phenotypes of both EC subdivisions likely depend on the combination of intrinsic organization and specific sets of inputs. We further suggest a reappraisal of the notion of EC as a layered input-output structure for the hippocampal formation. The denomination “entorhinal cortex (EC)” (Brodman’s area 28) is based on the fact that it is  enclosed by the rhinal (olfactory) sulcus. Interest in the EC arose around the turn of the 20th century when Ramón y Cajal, described a peculiar part of the posterior temporal cortex that was strongly connected to the hippocampus by way of the temporo-ammonic tract  or parahippocampal cortex . In view of a confusing nomenclature that has developed over the years to describe these different projection systems  make up the other main principal cell type in layer II of the MEC. PCs are typically calbindin-positive, although a few reelin-positive PC have been described , somatostatin (SOM) and 5HT3a expressing cells , comprises mainly large pyramidal neurons that are unequally distributed along the extent of both MEC and LEC. Cells in layer Vb appear smaller, more uniform in soma size and are more densely packed than their counterparts in layer Va . Whereas information about the postsynaptic targets of these cortical inputs is sparse, projections from the retrosplenial cortex (RSC) to MEC layer V target, among others, spiny pyramidal neurons that issue axons to superficial layers  of EC. The connectional scheme as presented here Figure  assumes ex vivo brain preparation, olfactory stimulation resulted in a sequential activation in LEC, hippocampus and MEC, followed by LEC (Biella and de Curtis, If correct, the connectional data strongly argue that differences in cortical inputs form a main feature underlying the phenotypic differences between LEC and MEC. However, we have not yet included the potential differences between LEC and MEC in local inhibitory architecture, as suggested by the yet sparse data on layer II. One additional feature of the proposed scheme needs to be discussed. The overarching strict reciprocal topology of the entorhinal-CA1-subicular network predicts that inbound information will be reciprocated with outbound information. It is exactly this last prediction, which is not supported by data. Admittedly, the available data are sparse, but the data obtained in the few studies in which this input-output dogma was addressed point to another direction. In one study in the cat, EEG recordings in freely behaving animals indicated a functional separation between LEC and MEC, where LEC is coupled to the olfactory domain, whereas MEC is coupled to the hippocampus (Boeijinga and Lopes da Silva, All authors contributed to the discussions that formed the foundation of the manuscript and contributed to the writing of the manuscript and to figures. All figures with exception of 1A were made by BJ. MPW supervised the process and wrote the final version of the manuscript. All authors approved this final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oxidative damage triggers extensive repair in gametes and thereafter in the zygote but it results in clinically relevant damage when affecting the maturation of the gametes chromatin, i.e. padlocking and epigenetic marking. It associates with defective DNA methylation and/or with oxidation of the methyl marks leading to derangement of gamete epigenetics, defects of chromatin condensation and aneuploidy. A proper feed to the one carbon cycle has the potential to stimulate the endogenous antioxidant defences, i.e. gluthatione synthesis, and to activate compensative homeostatic mechanisms restoring both the oxy-redox balance and DNA methylation, which are indeed strictly cross-regulated. This has been shown to produce measurable clinical improvements of male reproductive potential in pilot studies herein summarised. However, the effects of dietary habits and of supplementations are variable according to the individual genetic substrate, as genetic variants of several of the concerned enzymes occur with high frequency. Individual risk assessments and personalised interventions are still difficult to implement, in the meantime, a very varied diet may facilitate metabolic compensation in the majority of the cases. This review aims to report on the mechanisms of damage, on the opportunities to modulate the physiologic oxy-redox homeostasis by means of a varied diet or dietary supplements and on the open issues related to the genetic variability of the population. The energy supply to life on the Earth planet is mostly based on oxygen metabolism, and any healthy cell is well equipped to manage the possible damage from oxidising agents. Damage will occur only when the load of oxidative species exceeds the capacity of the cellular antioxidant mechanisms, which are largely dependent on the assumption of dietary substrates and micronutrients  and on tAmong the cellular species, gametes are the most sensitive to these imbalances due to their long maturation process in contact with the environment, which is particularly true for the male gametes. Evolution selected this type of process likely because gametes have the specific task to collect environment signals and to pass them to the offspring in the form of epigenetic marks . This leTherefore, it is worth putting more effort into the comprehension of oxy-redox homeostasis, the consequences of supporting interventions and the complex interaction with the environment, including feeding habits and genetic variants in the population.Oxidative molecular damage occurs in all cells but it is very likely to be repaired. Also spermatids, oocytes and thereafter zygotes possess powerful, although finite, DNA repair capacity . On the The methylation of DNA occurs at cytosine residues, parts of “cytosine-phosphate binding-guanosine” repeats called CpGs. The promoters of many genes contain areas enriched of CpG repeats called CpG islands. The extensive methylation of the CpG islands causes loss of affinity of the gene promoters for the transcription factors and results in gene silencing . In addiThe CpGs and the CpG islands are the main target of oxidative damage. The oxidation of the methylated cytosines within CpGs produces Hydroxy-methyl-Cytosine (OH-mC), which is the starting step for active demethylation  and may Oxidative stress-induced perturbations of DNA methylation also affects chromosome stability and segregation and may lead to aneuploidy of both sperm and oocytes. Meiosis requires full chromatin condensation to facilitate the process , which iIn summary, oxidative damage is likely to be of clinical relevance when and if resulting in derangement of epigenetic regulation as well as in perturbation of the gamete chromatin condensation phase, which does not leverage on repair mechanisms. The supportive interventions should be aimed at restoring both the full efficiency of the 1 carbon cycle (1CC) pathway, ensuring timed availability of activated methyl groups for DNA methylation, and proper reactivity by the intracellular antioxidant system, that will protect the epigenetic marks from misleading oxidation. Noteworthy, from a metabolic point of view, the 1CC and the antioxidant defences are a unique functional unit, i.e. two sides of the same coin .In physiologic conditions, in both somatic and germline cells, any excess of reactive oxygen species (ROS) activates removal by means of a variety of biochemical reactions and enzymes forming an anti-oxidant cascade that leverages on a wide list of mediators. These include enzymatic  and non-enzymatic  antioxidants . On top GSH is abundant in the cytosol, the mitochondria and the nuclei of all cells . It is aThe starting substrate for GSH de novo biosynthesis is Hcy, which is in turn the end-product of the 1CC , a tetrameric enzyme having pyridoxine (vit. B6) as the essential co-factor and binding Hcy by means of zinc fingers . It is hThe regulation of CBS activity, besides being the core of cell resistance to oxidative loads, is a paradigmatic example of how cell homeostasis works. The 1CC, being the pathway for adding the carbon units to molecules, ensures growth and differentiation of the cells and has a kind of metabolic priority. Within the pathways fed by the 1CC, DNA methylation has a further priority. The ability to defend the cell against oxidations is thereafter relevant only for cells that are duly growing with a positive balance of activated methyl groups (SAMe) to switch on GSH synthesis, otherwise the cell will be addressed to abortive apoptosis. The final effect is that the antioxidant defences will be active and efficient only in cells that are metabolically healthy.There is good consensus that oxidative damage to male gametes represents a relevant clinical issue , which oThe small effect on pregnancy rates is in part explained by the aggressiveness of oral antioxidants, forcing the cellular environment to the redox state, in front of the delicate metabolism of gametes that necessitate a perfect oxy-redox balance. Strong antioxidant cocktails may cause an excessive removal of oxygen free radicals essential for the regulation of several sperm functions: this may negatively affect capacitation and the acrosome reaction  and may It is anticipated that the reductive stress will interfere with the process of protamination and DNA synthesis. Both processes imply the resolution of the so-called supercoiling of DNA, which happens by extensive cut and paste work by the enzymes topoisomerase 2B (TOP2B) and poly-ADP-ribose-polymerase (PARP) . TOP2B gAnother problem is that, whatever the composition of antioxidant cocktails, it will have to contain a finite number of substances. Each of these substances will influence a finite number of oxy-redox reactions that, according to the dose of antioxidants administered, will be possibly imbalanced toward the reductive stress. Meantime, all the other oxy-redox reactions will remain unaffected. The final outcome is failure in equilibrating the intracellular environment and a possible reductive stress in selected pathways. This approach may be clinically advantageous any time the objective is the correction of a specific, well-known pathway but it is not suitable if the aim is to achieve a perfect oxy-redox balance and efficient DNA replication and protamination as required by gametogenesis.In summary, as the harmful effect of oxidative stress on gametes health is unquestionable and assumed the need to counteract the oxidative aggression in the sake of improved gametes quality, alternatives to the straight oral antioxidants administration are worthy to be explored.Once understood, the role of the 1CC in the activation of the physiologic antioxidant responses, it can be postulated that a robust support to the 1CC could result in improved DNA methylation, sustaining gametes maturation, and activation of the endogenous antioxidant system, configuring a physiologic and self-regulated antioxidant activity devoid of any risk of “reductive stress”. The 1CC is strictly dependant on dietary sources of methyl donors and of other essential substances, absolute or relative shortages may occur. In addition, a heavier endogenous (e.g. inflammation) or exogenous (e.g. pollutants) load of oxidative aggression may be responsible for a relative shortage also in individuals otherwise following a relatively healthy lifestyle.l-cystine) has the potential to feed GSH synthesis downstream to CBS and to allow the enlargement of the GSH-GSSG pool if it is needed.The list of dietary substances useful to sustain the 1CC is quite short and simple. Folates are to be included as the source for methyl groups for Hcy re-methylation. Quantities of riboflavin (vit. B2) and niacin (vit. B3) will support the conversion of folic acid to its active reduced form by the enzyme Methyl-Tetra-Hydro-Folate-Reductase (MTHFR). Vitamin B12 is then necessary to pass the methyl group from folates to Hcy. Finally, zinc, that has no body reservoirs and is strictly dependant on daily feeding , is alsoTM). Indeed betalaines have the property to stratify on plasma membranes where they act as suicide oxidation substrates to prevent the trigger of the lipoperoxidative cascade so to further aid sperm motility. This nutritional intervention was tested in male [A nutritional supplement containing all the substances necessary to support the 1CC  and the GSH production  was formulated and addressed to clinical trials. The supplement also contained small amounts of Vit. E (12 mg) as a stabilizer (to prevent premature oxidation of the cysteine donor) and betalain and quercetin of vegetal origin to protect membrane lipids  confirming that the new support strategy is effective in counteracting oxidative damage without negatively influencing (reductive stress) the nuclear decondensation. The study also recorded the pregnancy outcomes and, given the inclusion criteria, the results were encouraging. Eighteen couples (21 %) experienced a spontaneous pregnancy during the treatment, all of them ending with a live birth. The remaining 66 couples underwent a new ART attempt further resulting in 22 clinical pregnancies and in 15 live births. The overall clinical pregnancy rate (CPR) and live birth rate (LBR) were 47.6 and 39.3 %, respectively.The very first study was intended to confirm that: (i) The above formula does indeed exert antioxidant modulation in clinically significant amounts, and; (ii) The antioxidant gain does not generate any reductive stress as previously seen with oral antioxidants . A totalThe pregnancies seemed to be strongly related to the improvement of the sperm decondensation index Table  but the TM) [The intervention was then tested in comparison with a non-treatment group using the same supplement  or a simTM) . The secTM)  includedTM) , 46, agaTM) , 45 and Taken together, these data strongly support the idea that a tailored nutritional intervention in support of the 1CC and of GSH synthesis may have a fundamental impact on the reproductive prognosis of couples resistant to ART cycles and, in general, in infertile couples. The high rate of spontaneous pregnancies, not recorded in control groups, may in part result from a selection bias: repeated ART failures may have selected couples with a main metabolic problem. As a matter of fact, it has been demonstrated that morphologically normal spermatozoa with non-condensed chromatin may be found also in subjects with normal sperm parameters . TherefoThe strong relationship between the occurrence of a pregnancy and the improvement of sperm nuclear condensation in the male partner Table  may reprBased on the negative effects of direct antioxidants on sperm nuclear condensation , their iOnce the clinical opportunities arising from a tailored supplementation to the 1CC are understood, the next question is whether such supplementation would benefit the carriers of a deficient variant of the enzyme Methyl-Tetra-Hydro-Folate-Reductase (MTHFR). The enzyme variants are thermolabile, have a more rapid turn-over and exert reduced activity. The deficit of the MHTFR function according to the genotype is reported in Table Genetic variants of the MTHFR gene indeed have a high prevalence. The widely investigated C766T mutation has a variable prevalence according to ethnicity and geographical areas following a clear-cut north to south gradient with an incidence of the TT homozygotes in newborns ranging from <5 % in Northern Europe and Canada to >20 % in Southern Europe and Mexico . HoweverRecent metanalysis studies concluded for the MTHFR C677T , 53 and Thus, genetic variants may be a resistance factor to the treatment and a series of issues need to be clarified. Should we test the patients for MTHFR genotype? What do we do for homozygous subjects? Can we do better for heterozygous ones? Are there other genes involved?MTHFR is not the only enzyme of the 1CC occurring with a genetic polymorphism of functional consequences, rather the pathway is a concentrate of potential variants.The enzyme methionine synthase (MTR) catalyzes the methylation of Hcy to generate methionine. In case of defective activity there is lack of Hcy re-cycling. In addition, it is the only enzyme that uses 5-methyltetrahydrofolate and deficient activity also results in the trapping of cellular folate as 5-methyltetrahydrofolate , which bThe enzyme methionine synthase reductase (MTRR) is responsible of the reductive activation of MTR. Deficient MTRR activity causes hyperhomocysteinemia and all symptoms of B12 shortage even in presence of normal B12 levels. The defective MTRR variant A66G has been clinically linked with high Hcy and occurs with a frequency almost as high as MTHFR variants: according to a very large survey study in the USA almost half of the USA population was heterozygous for MTRR A66G with the highest prevalence in the non-Hispanic white ethnic group .The enzyme betaine homocysteine methyltransferase (BHMT) is responsible of Hcy methylation using betaine (trimethylglycine) as the methyl donor. A long list of single nucleotide polymorphisms (SNPs) has been described, all of them implicated with the occurrence of reproductive, neoplastic and degenerative diseases, but always within variable associations with other SNPs of the same gene or of other genes of metabolic enzymes configuring a kind of genetic puzzle. In example, the G742A mutation was associated with the occurrence of any neural tube defect (NTD) in a USA population but only in the subgroup of mothers supplemented with folic acid whereas in non-supplemented mothers it had no influence , suggestAs many as 150 clinically relevant mutations of CBS, the key enzyme for oxy-redox homeostasis, have been described . In mostA schematic map of the possible genetic blockades to the 1CC and the GSH synthesis is depicted in Fig. This complexity explains why a carrier of a heterozygous MTHFR defective gene may be either well compensated or seriously metabolically impaired depending on the status of the other enzymes. Accordingly, the practice to extensively test the MTHFR genetic status in subfertile men and women is a kind of non-sense. It is clear that any individual risk assessment would require testing a variety of SNPs from a variety of possibly involved genes, and we do not even know how many genes are potentially involved neither we are sure of which are the SNPs relevant to functional defects. Even less is known about the phenotype and function level resulting from their highly variable associations. In our opinion, it is a lot simpler to test blood Hcy in both partners of all subfertile couples deserving the MTHFR gene analysis to those resulting with frankly elevated Hcy levels, i.e. above 30 μmol/L. Those homozygous for low activity variants will benefit from the diagnostic information that unmasks a series of risk factors, e.g. thromboembolic risk and neurodegeneration. For all the others, any wild type or variant heterozygous state will not mark any difference, at least as long as we will remain unable to interpret a so complex picture.MTHFR variants are clearly linked to infertility and reproductive pathologies as well as to many life-shortening metabolic, degenerative and neoplastic diseases; thus, they should disappear within a few generations. Conversely, the variants can be found in a large part of every populations. How is this possible?The answer from the geneticists is clear and final: This is the best possible demonstration of the gene-environment interaction. The prevalence of defective MTHFR variants clearly increases following a North to South gradient, i.e. the higher the consumption of a Mediterranean type diet, the higher the frequency of the defective MTHFR enzyme . BesidesFor historical and cultural reasons, MTHFR is far better investigated but it is very likely that similar considerations may come true also for the other enzymes of concern. Thus, we should refer to all these functional variants of the metabolic enzymes as population variants, i.e. a genetic diversity creating strength in the concerned species, including humans. Apart a few exceptions, e.g. MTHFR C677T homozygosity and homocystinuria, none of these mutations constitutes a pathologic trait per se.The next point is to understand why Mediterranean diet compensates a lower MTHFR activity. The folates contained in many natural foods including meats, in all the fortified foods and in the vast majority of the dietary supplements are in the form of folic or folinic acid that necessitates MTHFR activity to be biologically effective. Lettuce, beans, tomatoes, broccoli and most of the vegetables typical of the Mediterranean diet contain the soluble and active form of folates methyl-tetrahydrofolate , i.e. thOn the other side meats are rich in the as well necessary B12 and zinc. Indeed, in spite of a potentially high intake of methyl-tetrahydrofolate, vegans develop fast onset high blood homocysteine. Thus, the more the diet is varied and comprehensive of all type of foods, the higher are the chances to achieve a balanced support of nutrients independently of the individual genetic inheritance of enzymes alleles and to enjoy well efficient 1CC and antioxidant defences.Healthy feeding habits and lifestyles are the basic requisite for a good health and for efficient reproductive function, but in many instances it may be not enough. The modern lifestyle makes the adherence to a well varied diet difficult and engaging. In addition, the toxic load from the environment is increasing and may peak in subjects with known  or unknown exposures. Needless to say, subjects homozygous for any defective variants of the enzymes or carrying an imbalanced pattern of heterozygosis for several of the enzymes are further exposed to relative dietary shortages.This is favoring an increasing haphazard use of dietary supplements aimed to compensate known or supposed defects, which causes several concerns about a possible over exposure to folic acid. Indeed every manufacturer of dietary supplements is including some folic acid by default. Thus, people consuming more than one supplement, which is very common , accumulReduced folates, namely 5-methyltetrahydrofolate, are the soluble/circulating and bioavailable form of this nutrient , i.e. methyltetrahydrofolate, which is the soluble, active form and that does not require MTHFR activation . The defThe above wide range support of micronutrients can be achieved with a better attention to the diet and is also suitable for the formulation of supplements (see Table Prospective clinical trials are needed to assess the actual benefits and limits of this approach, meanwhile, due attention to the diet so as to include as many as possible different food sources is highly recommended.In summary, there is a complex interaction between the environment, genes and our feeding habits. The individual outcome depends on a variety of genetic, epigenetic and behavioral factors. Clinically relevant damage is commonly occurring, mainly dependent on perturbations of the 1CC and has the deregulation of DNA re-arrangements among the most deleterious effects. The diet, in case associated to tailored supplementations, has the potential to adjust most of the imbalances in most of the subjects.Oxidative damage is clinically important because it has the potential to hamper gamete reproductive competence and to increase the aneuploidy rate;Oral antioxidants are powerful tools with strong pharmaceutical effects but misuse occurs easily; their chances to achieve a metabolic balance are negligible;The key to achieve an oxy-redox balance is the support to cellular homeostatic mechanisms: Full availability of micronutrients necessary to the 1CC favors the synthesis of GSH and the function of the endogenous antioxidant cascade resulting in measurable clinical gains;High attention should be given to the damage to the chromatin structure and to the epigenetic regulation of the gametes both in the diagnostic phase and in monitoring the effects of any interventions;The individual genetic status has a primary impact on the susceptibility to the damage, but risk assessment of the individual remains difficult to define. The screening of Hcy levels can be of help in selecting patients that benefit from a detailed genetic investigation;Although cheap and easy, folic acid is just a pre-vitamin and excessive consumption may create health problems. Food fortification programs should be further re-considered. The prescription of huge doses to subjects that do not metabolise it, e.g. MTHFR variant homozygous, is a questionable idea;Only a balanced and very varied diet has the potential to fulfill the needs in all subjects, once achieved dietary supplements can be of further help. Whatever stressed diet, including vegetarian, hyper-proteic and the like, has the potential to create pathologic imbalances;The activated forms of micronutrients are of greater help and their requirements can be fulfilled with tailored dietary regimens. They can also be provided by dietary supplements;Due attention to the metabolic issues and supportive interventions since the first referral have the potential to reduce the time to pregnancy of infertile couples and to increase the final cumulative success rate.Full understanding and proper management of all this aspects may have a fundamental impact on the reproductive prognosis as well as on global health. Pending further understanding of all the involved mechanisms, there are already good indications to be implemented in clinical practice:


Biochemical oscillations play an important role in maintaining physiological and cellular homeostasis in biological systems. The frequency and amplitude of oscillations are regulated to properly adapt to environments by numerous interactions within biomolecular networks. Despite the advances in our understanding of biochemical oscillators, the relationship between the network structure of an oscillator and its regulatory function still remains unclear. To investigate such a relationship in a systematic way, we have developed a novel analysis method called interaction perturbation analysis that enables direct modulation of the strength of every interaction and evaluates its consequence on the regulatory function. We have applied this new method to the analysis of three representative types of oscillators.The results of interaction perturbation analysis showed different regulatory features according to the network structure of the oscillator: (1) both frequency and amplitude were seldom modulated in simple negative feedback oscillators; (2) frequency could be tuned in amplified negative feedback oscillators; (3) amplitude could be modulated in the incoherently amplified negative feedback oscillators. A further analysis of naturally-occurring biochemical oscillator models supported such different regulatory features according to their network structures.Our results provide a clear evidence that different network structures have different regulatory features in modulating the oscillation frequency and amplitude. Our findings may help to elucidate the fundamental regulatory roles of network structures in biochemical oscillations.The online version of this article (10.1186/s12918-017-0472-7) contains supplementary material, which is available to authorized users. Oscillations are commonly observed phenomena in biological systems and perform crucial functions in regulating physiological or cellular processes . The beaOscillators appear to have different requirements for regulating the frequency and amplitude depending on their biological functions. Both frequency and amplitude of a circadian oscillator need to be regulated against fluctuations in order to maintain robust 24-h rhythms . In the Thus, how do these oscillators meet such different requirements for regulating the frequency and amplitude? Novak et al. classified biochemical oscillators into three classes: class I oscillators (delayed negative feedback oscillators), class II oscillators (amplified negative feedback oscillators), and class III oscillators (incoherently amplified negative feedback oscillators) . This clSuch an association between them could also be inferred from the previous study by Tsai et al. in which it was revealed that an interlinked positive and negative feedback structure outperforms a simple negative feedback structure in tuning the frequency of an oscillator . In addiSo far, the parameter perturbation method has been used to study the properties of oscillators. However, this method might not be adequate to analyze the network-level characteristics of oscillators. Because a parameter can represent various biological functions , perturbation of a parameter may not correspond to the variation of an interaction in the network. Moreover, the same molecular interaction can be represented in multiple ways . In these cases, the role of an interaction cannot be assessed independently through parameter perturbation analysis. A solution to such limitations could be to modulate the interactions rather than the parameters. For this purpose, we developed a novel perturbation strategy called the interaction perturbation method which directly modulates the strength of an interaction between two nodes in a network. By using this method, we found that a strong association exists between network structures and regulatory patterns of the frequency and amplitude of biochemical oscillators. In simple negative feedback oscillators, both the frequency and amplitude were found to be rarely modulated. In contrast, the frequency could be tuned in amplified negative feedback oscillators while the amplitude could be modulated in incoherently amplified negative feedback oscillators. Our analysis shows that different regulatory properties can emerge from different network structures of biochemical oscillators.In the three equations, the parameter ons  and nine possible types of interactions  Fig. . In all The simple negative feedback oscillator (simple NFO) has a negative feedback loop only Fig. . For theAccording to the classification by Novak et al., the above six models can be classified into three classes: the simple NFO belongs to class I oscillators; the activator-amplified NFO and the inhibitor-amplified NFO belong to class II oscillators; and all types of incoherently-amplified NFOs belong to class III oscillators .To investigate the regulatory patterns of the three-node oscillators, we performed perturbations by weakening each interaction by 1%, 2%, 4%, and 8%  and observed the changes in the frequency and amplitude. The interaction perturbation was implemented by multiplying a weakening factor to the element of Jacobian matrix that is to be perturbed during one period of oscillation. Fig. To represent the results quantitatively, we grouped the changes in the frequency and amplitude into three patterns: In pattern R, both the frequency and amplitude changed by less than 1%; in pattern F, either the frequency or the amplitude changed by more than 1% and the changes in the frequency were greater than the changes in the amplitude; in pattern A, either the frequency or the amplitude changed by more than 1% and the changes in the amplitude were greater than the changes in the frequency. Table The simple NFO showed the highest robustness to perturbations regardless of the types of perturbed interactions Fig.  and are In the activator-amplified NFO and inhibitor-amplified NFO, the change in the frequency was larger than the change in the amplitude. The results are shown in Fig. In contrast to the regulatory patterns observed in the activator-amplified NFO and the inhibitor- amplified NFO, all the incoherently-amplified NFOs showed moderate changes in the amplitude. In the type 1 incoherently-amplified NFO, the amplitude was slightly more adjustable to perturbations than the frequency while in the type 2 incoherently-amplified NFO, the amplitude was changed to a large extent Fig. . In the For the six 3-node oscillator models, the perturbation results obtained by weakening each interaction by 2%, 4%, or 8% had qualitatively the same regulatory patterns as those obtained by weakening each interaction by 1%  is robust to perturbations while for class II oscillators (the activator-amplified NFO and the inhibitor-amplified NFO), the frequency can be selectively regulated. In class III oscillators , the amplitude can be regulated. Based on these observations, we deduced the regulatory principle that the differences in network structures give rise to different regulatory patterns of the frequency and amplitude.Class I, class II and class III oscillators are structurally related to one another, and their structural differences arise from an added link. A class II oscillator can be formed by adding a link to the activator (X) or inhibitor (Y) of a class I oscillator. A class III oscillator can be formed by adding an incoherent link to a class I oscillator.This prompted us to assume that the added links could bring about changes to the regulatory patterns of the oscillators. To examine this idea, we performed mathematically controlled comparisons between structurally related oscillators.We developed three additional three-node oscillator models which contain one additional link to the backbone of a simple NFO. A self-activating positive feedback link was added to the activator (X) and inhibitor (Y) of the simple NFO to generate an activator-amplified NFO variant and an inhibitor-amplified NFO variant, both of which belong to class II oscillators. Adding Lxy to the simple NFO generated a variant of the type 1 incoherently-amplified NFO , which belongs to class III oscillators. Simulation of the simple NFO was performed with a representative parameter set suggested by Novak et al. . The parPerturbations on the oscillators were performed by weakening each interaction by 1% during one period of oscillation. A distinct regulatory pattern for each model could be identified despite the fact that the frequency and amplitude changed by less than 1% compared to the unperturbed cases in all four models Fig. . In the To explore whether the regulatory principle suggested here could also apply to naturally-occurring biochemical oscillator models, we performed analyses of nine well-known biochemical oscillator models which were constructed based on experimental data. For each model, perturbations were conducted by weakening each interaction by 1% during one-period of oscillation. The subsequent changes in the frequency and amplitude are shown in Fig. . These oscillators showed the highest robustness to perturbations: both the frequency and amplitude rarely changed in response to the perturbations. The sinus node model by Yanagihara et al. [The circadian rhythm model by Goldbeter , the cira et al. , the neua et al.  and the a et al.  can be ca et al.  and the a et al. , 23 thatIn summary, the regulatory principle suggested here in the three-node oscillator models could also apply to naturally-occurring biochemical oscillator models.Our analysis based on the interaction perturbation method revealed the regulatory principle that different network structures of biochemical oscillators give rise to different regulatory patterns of the frequency and amplitude; for class I oscillators, the frequency and amplitude are seldom regulated; for class II oscillators, the frequency is more adjustable than the amplitude; for class III oscillators, the amplitude is more adjustable than the frequency. The results of the mathematically controlled comparisons further demonstrated the reliability of this regulatory principle and its potential for application to naturally-occurring biochemical oscillator models.In systems biological studies, the parameter perturbation method has been widely used to investigate the relationship between network structures and their biological functions –29. In aetc) for the analysis of biochemical oscillators can provide meaningful insights into the nature of oscillators, but most of them require a certain level of expert knowledge on mathematics [The interaction perturbation method proposed in this study has several advantages over the parameter perturbation method. First, the result of an interaction perturbation analysis can be properly interpreted in the context of a biological network since the perturbation directly modulates a link of the network structure. Second, this method can provide a more pertinent comparison between different network structures by allowing the focus of the comparison to be placed on the difference of the interaction in the network, not on the indirect difference of the underlying biological process. If the network structures have the same number of nodes, the comparison can be performed more effectively between them as they have a Jacobian matrix of the same size (three-node networks have a Jacobian matrix of 3 by 3). Third, this method can simplify analysis procedures. Previously suggested methods  except for a few parameter sets where the amplitude was changed by more than 1% (pattern A). For class II oscillators, the frequency was adjustable for more than one third of the parameter sets (pattern F) whereas, for the others, the frequency was not changed (patterns R and A). For class III oscillators, the amplitude was adjustable for a relatively greater part of the parameter sets (pattern A) whereas, for the others, the amplitude was not changed (patterns R and F).For the activator-amplified NFO and inhibitor-amplified NFO, by perturbation of Lxx or Lxy, the frequency was adjusted whereas the perturbations did not significantly change the frequency or amplitude. In the incoherently amplified NFOs, only some kinds of interactions seem to be involved in modulation of the amplitude.Our analyses of naturally-occurring biochemical oscillator models showed that the regulatory principle suggested here may have applications in naturally-occurring biochemical oscillation models. A question then might arise as to what functional benefits can be derived from a particular network structure in naturally-occurring biochemical oscillators? Both frequency and amplitude of a circadian oscillator need to be regulated against fluctuations in order to maintain robust 24-h rhythms , 37. ThiFrom an evolutionary point of view, the fact that class II and class III oscillators are frequently found in natural biological examples can be considered as important evidence implicating that they might have some performance advantages over class I oscillators. This study suggests that such advantages might be found from the ability to tune the frequency in class II oscillators and the ability to regulate the amplitude in class III oscillators.Our study has the following limitations. Firstly, our study employed the Jacobian matrix which describes the interactions between state variables instead of employing the monodromy matrix  or Floquet multipliers  which have been widely used to determine the oscillatory properties of a system with a limit cycle. Therefore, our scope of analysis was largely confined to examining influences of interaction perturbation on the oscillatory properties. Secondly, interaction perturbation was performed during one cycle of oscillation because longer duration of perturbation destabilized the oscillation in most cases. Thirdly, our analysis of structurally related models may not be sufficient to investigate the general characteristics of each structure in greater detail since it was performed under the pre-defined parameter combinations.From the analyses based on the interaction perturbation method, we found a new regulatory principle that differences in network structures can give rise to different regulatory patterns of the frequency and amplitude. This finding could serve as a basis for further investigation into the underlying mechanism for the regulation of the frequency and amplitude in existing biochemical oscillators as well as for designing synthetic oscillators with a specific regulatory function.We constructed six representative oscillator models and generated random parameter sets for each model ensuring its sustained oscillation under the parameter sets. Then, we converted the interactions in the model into corresponding elements of the Jacobian matrix and performed perturbations of the elements. After the perturbations, we measured resulting changes of the frequency and amplitude. The analysis workflows are described in Fig. Each three-node oscillator model was described in terms of three coupled ODEs with the combinatorial use of mass action laws and Michaelis-Menten kinetics. The ODEs of the six 3-node oscillator models are provided in the Additional file Determining the parameter values constitutes an important process to create sustained oscillations. We chose to determine parameter values by extensive search of the parameter space because an analytical approach does not lend itself to dealing with a large number of parameters.Random parameter sets were generated for each three-node oscillator model by selecting parameters from an exponential distribution within the range of 0.001 to 1000, using the Latin hypercube sampling method . This raaij = ∂fi/∂xj, where i and j denote the row and column indices of the Jacobian matrix, respectively. A non-zero value of aijmeans that variable xj influences the evolution of variable xi; in other words, an interaction from node j to node i exists [A network topology shows clearly whether an interaction is activating or inhibiting. However, when this network is represented by the system of ODEs, the function of interaction is not easily identifiable. To represent an interaction as an algebraic object, the Jacobian matrix will be a reasonable choice since an element of the Jacobian matrix corresponds to an interaction. Because an element of the Jacobian matrix cannot be obtained directly from first-order ODEs, we differentiated the first-order ODEs with respect to time to generate second-order ODE systems  in the activator-amplified NFO variant are equal to the corresponding parameters of the simple NFO. Second, parameters associated with altered processes are free to assume any values. In the activator-amplified NFO variant, parameters related with the synthesis of X are assumed to have any values. Third, the parameters of the altered processes are determined by imposing constraints on the external behavior of the system. For the above three oscillation models, the following two constraints were imposed to determine the free parameters: (i) integration of the ODE models with the specified parameter sets have to be able to generate a limit-cycle oscillation; (ii) the frequency and amplitude of the oscillation have to be similar to those of the simple NFO.To determine the parameter sets for the activator-amplified NFO variant, the inhibitor-amplified NFO variant, and the type 1 incoherently-amplified NFO variant, we adopted a method for mathematically controlled comparisons proposed by Michael A. Savageau . First, After determination of the parameters, perturbations were performed in the same manner as in the conceptual three-node models. Then, the changes in the frequency and amplitude in the four oscillator models  were compared.