Assessing hemodynamics from the photoplethysmogram to gain insights into vascular age: a review from VascAgeNet.

The photoplethysmogram (PPG) signal is widely measured by clinical and consumer devices, and it is emerging as a potential tool for assessing vascular age. The shape and timing of the PPG pulse wave are both influenced by normal vascular aging, changes in arterial stiffness and blood pressure, and atherosclerosis. This review summarizes research into assessing vascular age from the PPG. Three categories of approaches are described: 1) those which use a single PPG signal (based on pulse wave analysis), 2) those which use multiple PPG signals (such as pulse transit time measurement), and 3) those which use PPG and other signals (such as pulse arrival time measurement). Evidence is then presented on the performance, repeatability and reproducibility, and clinical utility of PPG-derived parameters of vascular age. Finally, the review outlines key directions for future research to realize the full potential of photoplethysmography for assessing vascular age.

A Novel Physical Fatigue Assessment Method Utilizing Heart Rate Variability and Pulse Arrival Time towards Personalized Feedback with Wearable Sensors.

This paper proposes a novel method for physical fatigue assessment that can be applied in wearable systems, by utilizing a set of real-time measurable cardiovascular parameters. Daylength measurements, including a morning test set, physical exercise during the day, and an afternoon test set were conducted on 16 healthy subjects (8 female and 8 male). To analyze cardiovascular parameters for physical fatigue assessment, electrocardiography, pulse wave and blood pressure were measured during the test sets. The fatigue assessment questionnaire score, reaction time, countermovement jump height and hand grip strength were also measured and used as reference parameters. This study demonstrates that (i) the compiled test battery can selectively assess the rested vs. physically-fatigued states; (ii) the obtained linear support-vector machine, trained using the heart rate variability based parameter (F-score 0.842, accuracy 0.813) and pulse arrival time based parameter (F-score 0.875, accuracy 0.875) shows a promising ability to classify between the physically mildly fatigued and significantly fatigued states. Despite the somewhat limited study group size, the results of the study are unique and provide a significant advancement on the existing physical fatigue assessment methods towards a personalized and continuous real-time fatigue monitoring system with wearable sensors.

Serum Levels and Removal by Haemodialysis and Haemodiafiltration of Tryptophan-Derived Uremic Toxins in ESKD Patients.

Tryptophan is an essential dietary amino acid that originates uremic toxins that contribute to end-stage kidney disease (ESKD) patient outcomes. We evaluated serum levels and removal during haemodialysis and haemodiafiltration of tryptophan and tryptophan-derived uremic toxins, indoxyl sulfate (IS) and indole acetic acid (IAA), in ESKD patients in different dialysis treatment settings. This prospective multicentre study in four European dialysis centres enrolled 78 patients with ESKD. Blood and spent dialysate samples obtained during dialysis were analysed with high-performance liquid chromatography to assess uremic solutes, their reduction ratio (RR) and total removed solute (TRS). Mean free serum tryptophan and IS concentrations increased, and concentration of IAA decreased over pre-dialysis levels (67%, 49%, -0.8%, respectively) during the first hour of dialysis. While mean serum total urea, IS and IAA concentrations decreased during dialysis (-72%, -39%, -43%, respectively), serum tryptophan levels increased, resulting in negative RR (-8%) towards the end of the dialysis session (p < 0.001), despite remarkable Trp losses in dialysate. RR and TRS values based on serum (total, free) and dialysate solute concentrations were lower for conventional low-flux dialysis (p < 0.001). High-efficiency haemodiafiltration resulted in 80% higher Trp losses than conventional low-flux dialysis, despite similar neutral Trp RR values. In conclusion, serum Trp concentrations and RR behave differently from uremic solutes IS, IAA and urea and Trp RR did not reflect dialysis Trp losses. Conventional low-flux dialysis may not adequately clear Trp-related uremic toxins while high efficiency haemodiafiltration increased Trp losses.

Influence of gender on the QT interval variability and duration in different wake-sleep stages in non-sleep apneic individuals: Analysis of polysomnographic recordings.

INTRODUCTION: The aim of the study was to determine the influence of gender and sleep stages, especially rapid eye movement sleep (REM), on QT interval variability and duration in normal subjects. METHODS: Polysomnographic recordings of 24 male and 24 female patients without obstructive sleep apnea were analyzed. In each patient, the QT interval variability index (QTVI) and the corrected QT interval (QTc) values were calculated as means of 2 awake, 4 non-rapid eye movement sleep (NREM) and 3 REM episodes, 300s each. For the QTc calculation, five different correction formulas were used. RESULTS: Gender-related differences in the QT interval variability and duration were detected between all sleep stages (P<0.05). In males, mean values of QTVI while awake, in NREM and REM sleep were -1.1±0.2, -1.1±0.3, -1.3±0.2. In females, mean values of QTVI were -0.9±0.4, -0.9±0.4, and -1.1±0.3, respectively. No difference between sleep stages was detected in the mean values of QTVI and QTc in both groups (P>0.05). CONCLUSION: The results of our study demonstrate no significant overall impact of sleep stages on ventricular repolarization variability and duration during physiological sleep in both genders. We found gender differences in the mean values of QTVI and QTc during different sleep stages, which confirm that gender is a modulating factor of ventricular repolarization.

Microwave effect on diffusion: a possible mechanism for non-thermal effect.

In this study, we assume that microwave radiation affects hydrogen bonding between dipolar water molecules and through that diffusion in water at constant temperature. The experimental study was performed on the setup of two identical reservoirs filled with pure water and 0.9% NaCl solution and connected by a thin tube. Alterations of NaCl concentration in the reservoir initially filled with pure water were measured using the resistance of the solution as an indicator. The applied 450 MHz continuous-wave microwave field had the maximal specific absorption rate of 0.4 W/kg on the connecting tube. The standard deviation of water temperature in the setup was 0.02 °C during an experiment. Our experimental data demonstrated that microwave exposure makes faster the process of diffusion in water. The time required for reduction of initial resistance of the solution by 10% was 1.7 times shorter with microwave. This result is consistent with the proposed mechanism of low-level microwave effect: microwave radiation, rotating dipolar water molecules, causes high-frequency alterations of hydrogen bonds between water molecules, thereby affects its viscosity and makes faster diffusion.

Effect of transmural pressure on the estimation of arterial stiffness index from the photoplethysmographic waveform.

The aim of this study was to find the effect of transmural pressure on the determination of the photoplethysmographic (PPG) waveform arterial stiffness index (PPGAI). The study was conducted on 51 subjects without diagnosis of cardiovascular disease, aged between 24 and 74 years. The relation between the transmural pressure, which is the difference between the arterial blood pressure and the PPG sensor contact pressure, and the PPGAI was determined. PPG, beat-to-beat blood pressure, and sensor contact pressure signals were recorded from the index, middle, and ring finger. The PPG sensor contact pressure of the index finger was increased from 20 to 120 mmHg. The aortic augmentation index (AIx@75) was estimated with a SphygmoCor device as a reference. High correlation coefficients r = 0.79 and r = 0.83 between PPGAI and AIx@75, and low PPGAI standard deviations were observed at the transmural pressures of 10 and 20 mmHg, respectively. Transmural pressure of 20 mmHg can be considered suitable for the PPG signal registration and PPGAI calculation for the assessment of arterial stiffness. In summary, the contact pressure of the sensor should be selected according to theblood pressure of the subject finger in order to achieve the transmural pressure suitable for the assessment of PPGAI and arterial stiffness.

New photoplethysmographic signal analysis algorithm for arterial stiffness estimation.

The ability to identify premature arterial stiffening is of considerable value in the prevention of cardiovascular diseases. The "ageing index" (AGI), which is calculated from the second derivative photoplethysmographic (SDPPG) waveform, has been used as one method for arterial stiffness estimation and the evaluation of cardiovascular ageing. In this study, the new SDPPG analysis algorithm is proposed with optimal filtering and signal normalization in time. The filter parameters were optimized in order to achieve the minimal standard deviation of AGI, which gives more effective differentiation between the levels of arterial stiffness. As a result, the optimal low-pass filter edge frequency of 6 Hz and transitionband of 1 Hz were found, which facilitates AGI calculation with a standard deviation of 0.06. The study was carried out on 21 healthy subjects and 20 diabetes patients. The linear relationship (r = 0.91) between each subject's age and AGI was found, and a linear model with regression line was constructed. For diabetes patients, the mean AGI value difference from the proposed model y AGI was found to be 0.359. The difference was found between healthy and diabetes patients groups with significance level of P < 0.0005.

Optical Real-Time Cardiorenal Toxin Uric Acid Measurement During Hemodialysis Using a Miniaturized Optical Sensor.

Patients with chronic kidney disease (CKD) are at higher cardiovascular risk than the general population. Cardiovascular diseases, vascular calcification among them, are the leading cause of death in these patients. Factors influencing vascular calcification are oxidative stress, inflammation, and accumulation of uremic toxins during CKD. Uric acid is a cardiorenal toxin that accumulates in the case of kidney malfunction. The primary therapy for replacing kidney function and removing toxins from end-stage renal disease patients is hemodialysis. Effective removal of toxins can be estimated by blood or dialysate lab analysis or optical monitoring. In this study, the authors tested a miniaturized optical sensor for monitoring uric acid levels and removal for the first time in a more extensive clinical study, including Hemodialysis (HD) and Post-dilutional online hemodiafiltration (HDF) procedures with different settings in Tallinn, Estonia. The results (Mean±SD, Lab vs. Sensor) of the uric acid concentration 57.20±34.05 vs. 57.22±33.09 µmol/L, reduction ratio 68.72±10.91 vs. 67.89±12.48 %, and total removed amount 7.00±2.10 vs. 7.33±2.29 mmol did not differ significantly from the values obtained from the clinical laboratory (p<0.05).Clinical Relevance-During this study, a miniaturized optical sensor was tested for the first time in the clinic in different dialysis settings. The results confirm that the sensor is reliable for regularly monitoring cardiorenal toxin uric acid removal during hemodialysis.

Time-averaged concentration estimation of uraemic toxins with different removal kinetics: a novel approach based on intradialytic spent dialysate measurements.

Background: Kt/Vurea is the most used marker to estimate dialysis adequacy; however, it does not reflect the removal of many other uraemic toxins, and a new approach is needed. We have assessed the feasibility of estimating intradialytic serum time-averaged concentration (TAC) of various uraemic toxins from their spent dialysate concentrations that can be estimated non-invasively online with optical methods. Methods: Serum and spent dialysate levels and total removed solute (TRS) of urea, uric acid (UA), indoxyl sulphate (IS) and ß2-microglobulin (ß2M) were evaluated with laboratory methods during 312 haemodialysis sessions in 78 patients with four different dialysis treatment settings. TAC was calculated from serum concentrations and evaluated from TRS and logarithmic mean concentrations of spent dialysate (MlnD). Results: Mean (± standard deviation) intradialytic serum TAC values of urea, UA, ß2M and IS were 10.4 ± 3.8 mmol/L, 191.6 ± 48.1 µmol/L, 13.3 ± 4.3 mg/L and 82.9 ± 43.3 µmol/L, respectively. These serum TAC values were similar and highly correlated with those estimated from TRS [10.5 ± 3.6 mmol/L (R 2 = 0.92), 191.5 ± 42.8 µmol/L (R 2 = 0.79), 13.0 ± 3.2 mg/L (R 2 = 0.59) and 82.7 ± 40.0 µmol/L (R 2 = 0.85)] and from MlnD [10.7 ± 3.7 mmol/L (R 2 = 0.92), 191.6 ± 43.8 µmol/L (R 2 = 0.80), 12.9 ± 3.2 mg/L (R 2 = 0.63) and 82.2 ± 38.6 µmol/L (R 2 = 0.84)], respectively. Conclusions: Intradialytic serum TAC of different uraemic toxins can be estimated non-invasively from their concentration in spent dialysate. This sets the stage for TAC estimation from online optical monitoring of spent dialysate concentrations of diverse solutes and for further optimization of estimation models for each uraemic toxin.

Establishing best practices in photoplethysmography signal acquisition and processing.

Photoplethysmography is now widely utilised by clinical devices such as pulse oximeters, and wearable devices such as smartwatches. It holds great promise for health monitoring in daily life. This editorial considers whether it would be possible and beneficial to establish best practices for photoplethysmography signal acquisition and processing. It reports progress made towards this, balanced with the challenges of working with a diverse range of photoplethysmography device designs and intended applications, each of which could benefit from different approaches to signal acquisition and processing. It concludes that there are several potential benefits to establishing best practices. However, it is not yet clear whether it is possible to establish best practices which hold across the range of photoplethysmography device designs and applications.

Treatment with Paracetamol Can Interfere with the Intradialytic Optical Estimation in Spent Dialysate of Uric Acid but Not of Indoxyl Sulfate.

Optical online methods are used to monitor the haemodialysis treatment efficiency of end stage kidney disease (ESKD) patients. The aim of this study was to analyse the effect of the administration of UV-absorbing drugs, such as paracetamol (Par), on the accuracy of optical monitoring the removal of uremic toxins uric acid (UA) and indoxyl sulfate (IS) during standard haemodialysis (HD) and haemodiafiltration (HDF) treatments. Nine patients received Par in daily dosages 1−4 g for 30 sessions. For 137 sessions, in 36 patients the total daily dosage of UV-absorbing drugs was less than 500 mg, and for 6 sessions 3 patients received additional UV-absorbing drugs. Par administration slightly affected the accuracy of optically assessed removal of UA expressed as bias between optically and laboratory-assessed reduction ratios (RR) during HD but not HDF employing UV absorbance of spent dialysate (p < 0.05) at 295 nm wavelength with the strongest correlation between the concentration of UA and absorbance. Corresponding removal of IS based on fluorescence at Ex280/Em400 nm during HD and HDF was not affected. Administration of UV-absorbing drugs may in some settings influence the accuracy of optical assessments in spent dialysate of the removal of uremic solutes during haemodialysis treatment of ESKD patients.

Association between ventricular repolarization and main cardiovascular risk factors.

OBJECTIVE: To assess the association between QT interval, QT dispersion and main cardiovascular risk factors in Tallinn women population aged 50-69 years. DESIGN: A random sample of Tallinn population, 302 women underwent 12-lead rest ECG, echocardiography and laboratory tests. Corrected QT interval and QT dispersion were calculated. RESULTS: The significant correlation coefficients for corrected QT interval and dispersion were observed with systolic, diastolic, pulse pressure, blood pressure value grade, heart rate, left ventricular mass index, and total cardiovascular risk grade. According to the data of multiple logistic regression, risk factors independently associated with prolonged QTc and QTD were arterial hypertension: OR 2.69, p < 0.001 and OR 3.29, p < 0.001, pulse pressure ≥ 60 mm Hg: OR 2.62, p < 0.001 and OR 3.04, p < 0.001, age > 65 years: OR 2.44, p < 0.001 and OR 1.91, p < 0.01, family history of cardiovascular disease: OR 2.02, p < 0.01 and OR 2.48, p < 0.001, left ventricular hypertrophy: OR 2.78, p < 0.001 and OR 5.29, p < 0.001. Multivariable linear regression data showed an independent association between corrected QT interval, QT dispersion and systolic blood pressure: ß = 0.32, p < 0.001 and ß = 0.39, p=0.003, pulse pressure: ß = 0.21, p = 0.004 and ß = 0.27, p < 0.001, blood pressure value grade: ß = 0.35, p < 0.001 (for QT dispersion only), heart rate: ß = 0.21, p < 0.001 and ß = -0.19, p = 0.001, left ventricular mass index: ß = 0.23, p < 0.001 and ß = 0.27, p < 0.001. CONCLUSION: In Tallinn women population, electrocardiographic parameters reflecting ventricular repolarization are associated with systolic blood pressure, pulse pressure, blood pressure value grade, heart rate and left ventricular hypertrophy.

In-Vitro Investigation of Flow Profiles in Arteries Using the Photoplethysmograph.

The flow profile in the artery reflects the health status of the vessel and generally the arterial system. The aim of this pilot study was to investigate in-vitro the effect of flow profiles on reflective photoplethysmography (PPG) signals at different steady state flow rates and levels of vessel constrictions. A simplified model of an arterial system was built, consisting of a steady state flow gear pump, PVC vinyl tubing, reservoir and a clamp with a micrometer gauge. The blood mimicking fluid (2.5% India ink and water solution) was pumped through the model. It was found that the waveforms of the PPG signals fluctuate irregularly and the magnitude of the frequency components was increased below 60 Hz in cases of turbulent flow (Re = 2503). These preliminary results suggest that PPG could be the basis for new technologies for assessing the profile of the blood flow in the artery. Future studies have to be carried out with pulsatile flow and more complex models that are more similar to the human arterial system.Clinical Relevance- The PPG signal reflects changes in the flow profile caused by the stenotic rigid vessel.

Aortic Augmentation Index is Dependent on Bodyside in Healthy Young Subjects.

INTRODUCTION: Aortic augmentation index (AIx) is a commonly used measure to evaluate the arterial stiffness of large elastic arteries. It has been used as an indicator for cardiovascular risk in clinical practice. AIM: To evaluate the difference in the aortic AIx assessed from the left and the right hand in a group of healthy young adults using SphygmoCor and Arteriograph devices. METHODS: 32 subjects were enrolled in this study (27 ± 7 years), 16 male and 16 female volunteers participated. Equally, half of the gender groups were left-handed and another half right-handed. RESULTS: It was found that the aortic AIx values assessed from the pressure waveforms of the right and the left hand are different and significantly higher in the left hand. Using a SphygmoCor device, the mean difference between the aortic AIx values from the right and the left hand among the whole study group was found - 4.78 ± 4.31% and using an Arteriograph the aortic AIx values were - 3.92 ± 3.90%. Aortic AIx values assessed from the right and the left hand were linearly related to each other for both devices. Moreover, it was found that the values of the aortic. CONCLUSIONS: AIx are independent of the subject's handedness. It has to be pointed out that subjects who cannot be subjected to assessment of the aortic AIx from one side of the body could have different AIx values estimated from the recorded pressure waveform from the other bodyside.

Pulse wave velocity and its gender-related associations with cardiovascular risk factors in a high cardiovascular risk population.

INTRODUCTION: Associations found between pulse wave velocity (PWV) and cardiovascular risk factors (CVrF) are diverse. We aimed to evaluate whether differences in PWV and its associations with CVrF in a high cardiovascular risk population exist between genders and between the whole population (WHgr) and groups of apparently healthy (AHgr) and those of hypertensive, obese or diabetics (Rgr). MATERIAL AND METHODS: Pulse wave velocity measured by Arteriograph was investigated in 805 adults aged 20-65, randomly selected from the Tallinn Population Register. RESULTS: Pulse wave velocity was the highest in Rgr and no differences were found between genders of the same group. In women of WHgr and AHgr age and SBP with addition of BMI and apolipoprotein B (ApoB) were associated with 54% and 48%, and without ApoB in Rgr with only 30% of PWV values. In men aged ≥ 50 of WHgr with elevated SBP odds ratios for increased PWV were 25.3 and 3.5, in Rgr 21.2 and 2.2, in those aged ≥ 50 AHgr 28.4. In women aged ≥ 50 of WHgr with elevated SBP and diabetes odds ratios were 5.5, 4.9 and 4.0, in Rgr with elevated SBP and diabetes 3.6 and 3.7, in those aged ≥ 50 AHgr 29.3. CONCLUSIONS: The associations of ApoB and BMI with PWV and diabetes with elevated PWV indicative of increased aortic stiffness were unique for women. Aging and SBP were related to PWV even in AHgr, although age ≥ 50 years in Rgr women and normal SBP in AHgr were not associated with elevated PWV.

QT interval variability index and QT interval duration during different sleep stages in patients with obstructive sleep apnea.

OBJECTIVES: The aim of the study was to investigate the impact of obstructive sleep apnea (OSA) on the QT interval variability and duration in patients during different sleep stages. METHODS: Polysomnographic recordings of 28 (13 male, 15 female) patients with OSA and 30 (15 male, 15 female) patients without OSA were analyzed. The QT interval variability index (QTVI) and the corrected QT interval (QTc) analyses were performed using two awake, 3-4 non-rapid eye movement (NREM) and three rapid eye movement (REM) sleep episodes (each 300 s). The Bazett formula, linear, and parabolic heart rate correction formulas with two separate α values were used. RESULTS: QTVI was statistically higher in OSA than in non-OSA patients for males while awake (awake -0.7 ± 0.3 vs -1.2 ± 0.2, p = 0.001; NREM ‒0.9 ± 0.4 vs -1.1 ± 0.3, p = 0.110; REM ‒1.1 ± 0.3 vs -1.3 ± 0.2, p = 0.667) and for females in all wake-sleep stages (awake -0.3 ± 0.7 vs -0.9 ± 0.5, p = 0.001; NREM ‒0.3 ± 0.5 vs -0.8 ± 0.4, p = 0.002; REM -0.3 ± 0.5 vs -1.0 ± 0.4, p < 0.001). QTVI was significantly higher during awake compared to sleep stages in OSA males (p < 0.05); no difference between wake-sleep stages was found in females (p > 0.05). Significant gender differences in QTVI existed in OSA patients during sleep (p < 0.05) but not while awake. No significant differences in QTc between patients groups were observed. CONCLUSIONS: OSA is associated with increased QT variability. REM sleep per se does not increase QTVI. In OSA patients, QTVI might be a more useful measure to detect ventricular repolarization abnormality than measures of QTc.

Smart photoplethysmographic sensor for pulse wave registration at different vascular depths.

The aim of this paper is to propose a smart optical sensor for cardiovascular activity monitoring at different tissue layers. Photoplethysmography (PPG) is a noninvasive optical technique for monitoring mainly blood volume changes in the examined tissue. However, different important physiological parameters, such as oxygen saturation, heart and breathing rate, dynamics of skin micro-circulation, vasomotion activity etc., can be extracted from the registered PPG signal. The developed sensor consists of 32 light emitting sources with four different wavelengths, which are located to the four different distances from four photo detectors. Compared to the existing sensors, the system enables to select the optimal LED (light emitting diode) and photo detector couple in order to obtain the pulse wave signal from the interested blood vessels with the highest possible signal to noise ratio. In this study, the designed PPG sensor was tested for the pulse wave registration from radial artery. The highest efficiency and signal to noise ratio was achieved using infrared LED (940 nm) and photo-diode pair.

QT Interval Variability Index and QT Interval Duration in Different Sleep Stages: Analysis of Polysomnographic Recordings in Nonapneic Male Patients.

The aim of the study was to determine whether different sleep stages, especially REM sleep, affect QT interval duration and variability in male patients without obstructive sleep apnea (OSA). Polysomnographic recordings of 30 patients were analyzed. Beat-to-beat QT interval variability was calculated using QTV index (QTVI) formula. For QTc interval calculation, in addition to Bazett's formula, linear and parabolic heart rate correction formulas with two separate α values were used. QTVI and QTc values were calculated as means of 2 awake, 3 NREM, and 3 REM sleep episodes; the duration of each episode was 300 sec. Mean QTVI values were not statistically different between sleep stages. Therefore, elevated QTVI values found in patients with OSA cannot be interpreted as physiological sympathetic impact during REM sleep and should be considered as a risk factor for potentially life-threatening ventricular arrhythmias. The absence of difference of the mean QTc interval values between NREM and REM stages seems to confirm our conclusion that sympathetic surges during REM stage do not induce repolarization variability. In patients without notable structural and electrical remodeling of myocardium, physiological elevation in sympathetic activity during REM sleep remains subthreshold concerning clinically significant increase of myocardial electrical instability.

Pulse wave registration from radial artery using photoplethysmographic method.

This pilot study was aimed to investigate the possibilities to use the photoplethysmographic (PPG) method for the pulse wave registration from radial artery. The PPG sensor with different separation distances between light emitting diodes (LED) and photodiode was built. The PPG signal registration was carried out at the locations with two different depths of artery and at the locations without large blood vessels under the sensor. In addition, two different forces were applied on the sensor in order to decrease the blood volume in underlying tissue and lower the pulsations that originates from smaller vessels. As a result, it was found that the artery was possible to locate under the sensor, where the value of DC component is minimal. Furthermore, the pressure has to be applied on sensor and optimal separation distance has to be selected between LED and photodiode for the pulse wave registration from radial artery. Further studies and improvements of the sensor are needed.

The effect of local cold and warm exposure on index finger photoplethysmographic signal waveform.

The study was carried out in order to analyse the changes in photoplethysmographic (PPG) signal waveform, which are caused by cold and warm stimulation. The study was carried out on 7 subjects. The right hand was immersed in cold and warm water up to the wrist during the experiment. The PPG signal was registered from right hand index finger. As a reference, the temperatures of index finger and pulse transit time (PTT) values were measured. A correlation of r = -0.70 was found between normalised slope of the PPG signal and the subject's age. The normalised slope increased noticeably due to the cold and decreased due to the warm water stimulation. It was opposite to the expected results with PTTs and the behaviour has to be investigated in future studies.