During chronic kidney disease, the progressive deterioration of renal function induces several biological/clinical dysfunctions, including enhancement of synthesis of inflammation/oxidative stress mediators. Impaired renal function is an independent cardiovascular risk factor; indeed, cardiovascular complications dominate the landscape of both chronic kidney disease and end-stage renal disease. The aim of this study is to explore the correlation between the global oxidative balance in hemodialysis patients and both inflammatory markers and cardiovascular events. Using photometric tests, this study explored plasmatic oxidative balance in 97 hemodialysis patients compared to a healthy population. In the hemodialysis patients, we showed that oxidative stress values were significantly lower than in controls while effectiveness in the antioxidant barrier was significantly increased in the hemodialysis group. Furthermore, we highlighted a strong correlation between oxidative index and blood levels of C-reactive protein. When patients were divided into two groups based on previous cardiovascular events, we found that subjects with previous cardiovascular events had higher values of both oxidative stress and antioxidant barrier than patients without cardiovascular events. Our results indicated that in hemodialysis patients, the clinical and prognostic significance of oxidative status is very different from general population. As cardiovascular complications represent a strong negative factor for survival of hemodialysis patients, the research of new cardiovascular risk biomarkers in these patients takes on particular importance in order to translate them into clinical practice/primary care. Chronic kidney disease (CKD) is a major public health problem worldwide, and its main consequences include loss of renal function leading to end-stage renal disease (ESRD), increased risk of cardiovascular disease (CVD), significant increase in morbidity and mortality, and a decrease in health-related quality of life –3. A recDuring CKD, the progressive deterioration of kidney function has important systemic effects due to its central role in body homeostasis and induces several biological and clinical dysfunctions including alteration in cellular energetic metabolism, change in nitrogen input/output, protein malnutrition, resistance to insulin, and considerable enhancement of synthesis of inflammation/oxidative stress mediators. Inflammatory markers such as C-reactive protein and cytokines increase with renal function deterioration suggesting that CKD is a low-grade inflammatory process  comparabImpaired renal function is an independent cardiovascular risk factor; indeed, cardiovascular complications dominate the landscape of both CKD and ESRD . The comThe aim of this study is to explore the global oxidative balance in ESRD hemodialysis (HE) patients compared to a healthy population. Since inflammatory stress and cardiovascular complications represent two very frequent conditions in hemodialysis subjects, the correlation between the oxidative parameters and both inflammatory markers and cardiovascular events was also investigated.The study involved 97 patients older than 18 years of both sexes with ESRD on regular hemodialysis therapy for at least 6 months  recruited from the Department of Nephrology, Dialysis and Transplantation, Kidney and Transplantation Research Center, Annunziata Hospital of Cosenza , between August 2017 and December 2017. The hemodialysis prescription consisted of 4 hourly, thrice weekly sessions, using polysulfone membrane and bicarbonate-buffered dialysate. The blood flow rate was 300 mL/min and low molecular weight heparin was used as anticoagulant. Hemodialysis adequacy was determined by using Kt/V formula. 2), while blood pressure was measured in the sitting position using an automatic sphygmomanometer (Microlife BP A2). The biochemical measurements included glucose, total cholesterol, triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), HbA1c, uric acid (UA), and creatinine levels. Blood tests also included the evaluation of prooxidant, and antioxidant status. Exclusion criteria included a family history of ESRD or an estimated glomerular filtration rate (eGFR) lower than 60 mL/min/1.73 m2, calculated by CKD-EPI equation [As controls for patient group, a recruitment campaign focused on the staff of the University of Calabria within the frame of the prevention and protection program adopted by the same university. In particular, 95 healthy volunteers age- and sex- matched with the ESRD group  were recruited between August 2017 and December 2017. They underwent through a structured interview during which sociodemographic, anthropometric, and clinic information was collected. The lifestyle was evaluated using self-administered questionnaires, which included items on smoking, exercise habits, and drinking habits. Anthropometric measurements included measurements of weight and height. Body mass index (BMI) was calculated as weight (kg)/height (mequation .All subjects (patients and controls) were studied in the morning and in a fasting state. Blood samples were taken from the antecubital vein and immediately centrifuged (2500 g for 15 min at 4°C), and the plasma obtained was stored at 4°C until measurements (maximum 6 hours of venous blood collection). Standard blood tests were performed at the clinical laboratory of Annunziata Hospital, Cosenza; prooxidant and antioxidant status were determined at the Analysis and Research on Oxidative Stress Laboratory (LARSO) of the University of Calabria.μmol/L of the reduced ferric ions.Oxidative stress and biological antioxidant potential determination were performed by using photometric measurement kits and a free radical analyzer system provided with spectrophotometric device reader . The d-ROM test helps to determine the oxidant ability of a plasma sample measuring the presence of reactive oxygen metabolite derivatives, in particular, hydroperoxides (oxidative index). Results are expressed in Carratelli units . The BAP test provides an overall measure of the biological antioxidant potential measuring the blood concentration of antioxidants  capable of reducing the iron from ferric to the ferrous form (antioxidant barrier). Results are expressed in t-test. The correlation between continuous variables was assessed using Pearson's coefficient. Data have been analyzed using R statistical language (http://www.r-project.org/).Differences between groups were examined using the independent sample Our study population consists of 97 hemodialysis patients and 95 healthy volunteers as control group. P value = 0.002). Moreover, ESRD patients also showed a greater effectiveness in the antioxidant barrier than CTR subjects . The relationship between oxidative index and antioxidant barrier values showed a borderline correlation  had higher values of both oxidative stress and antioxidant barrier than patients without cardiovascular events  were not related to both oxidative index and antioxidant barrier efficacy. Concerning smoking habits, it has not been possible to analyze any correlations given the small number of smoking subjects. Interestingly, when HD patients were stratified according to previous CVD events, we detected that subjects with previous acute myocardial infarction (CVD+, Our research focused on the evaluation of the overall redox state on a significant hemodialysis population. Our results clearly showed that the hemodialysis patients presented oxidative stress values significantly lower and antioxidant barrier effectiveness significantly higher compared to healthy controls. Despite the good plasmatic redox status found in all hemodialysis patients, we highlight a strong correlation between oxidative index and C-reactive protein blood levels. In addition, we reported that, in our cohort of hemodialysis patients, subjects with previous acute myocardial infarction had higher values of both oxidative stress and antioxidant barrier respect to patients without cardiovascular events. It is noteworthy that these results have been realized using noninvasive method to detect total plasma redox balance, which is of particular importance when analyzing the frail hemodialysis patients. Indeed, to detect plasmatic redox status, we utilized two simple methods widely used in recent years, the d-ROM and the BAP tests –20. The Several authors reported a profound imbalance between oxidants and antioxidants in CKD . The balIn our cohort of hemodialysis patients, we detected oxidative stress values significantly lower compared to healthy controls. As index of oxidative stress, we did not analyze the oxidative damage of single macromolecules but the plasmatic oxidant ability by measuring the presence of reactive oxygen metabolite derivatives, in particular, hydroperoxides. Actually, the most direct approach for lipid peroxidation evaluation is the quantification of the primary products, hydroperoxides, rather than the secondary or end products derived from hydroperoxides such as malondialdehyde. Plasmatic hydroperoxides are a sensitive and specific index that the presence of oxidative stress in vivo providing a global assessment of the imbalance induced by the alteration of the individual parameters . Indeed,Concerning the antioxidant systems, literature data are really complex to discern. Most of the studies in CKD patients have analyzed expression/activity of antioxidant enzymes with conflicting results , 27. In CKD is a low-grade, chronic, systemic inflammation with considerable enhancement of synthesis of inflammation mediators such as C-reactive protein and cytokines . LiteratIn recent decades, numerous studies and meta-analyses have highlighted that impaired renal function is an independent cardiovascular risk factor; indeed, cardiovascular complications dominate the landscape of both CKD and ESRD . TraditiOur results suggest that in HD patients, the clinical and prognostic significance of oxidative status associated with cardiovascular risk factors is very different from the general population. Although a direct causality cannot be inferred from such kind of correlative investigations, our data provide an important contribution to understanding the cross-talk between oxidative imbalance and cardiovascular risk in CKD also representing a point of departure to address further investigation. As cardiovascular complications represent a strong negative factor for hemodialysis subject survival, the preservation of an optimal global redox balance through effective methods may be considered as a potential target for therapies aimed at preventing cardiovascular complications during CKD progression.


This clinical case report presents synchronous physiological data from an individual in whom a spontaneous vasovagal reaction occurred without syncope. The physiological data are presented for three main phases: Baseline (0–200 s), vasovagal reaction (200–600 s), and recovery period (600–1200 s). The first physiological changes occurred at around 200 s, with a decrease in blood pressure, peak in heart rate and vastus lateralis tissue oxygenation, and a drop in alpha power. The vasovagal reaction was associated with a progressive decrease in blood pressure, heart rate and cerebral oxygenation, whilst the mean middle cerebral artery blood flow velocity and blood oxygen saturation remained unchanged. Heart rate variability parameters indicated significant parasympathetic activation with a decrease in sympathetic tone and increased baroreflex sensitivity. The total blood volume and tissue oxygenation index (TOI) dropped in the brain but slightly increased in the vastus lateralis, suggesting cerebral hypoperfusion with blood volume pooling in the lower body part. Cerebral hypoperfusion during the vasovagal reaction was associated with electroencephalography (EEG) flattening  followed by an EEG high-amplitude “slow” phase . The subject developed signs and symptoms of pre-syncope with EEG flattening and slowing during prolonged periods of symptomatic hypotension, but did not lose consciousness. Vasovagal reactions include arterial vasodilation and bradycardia as mechanisms that may precipitate a syncopal response ; defined3 in which he may have felt confined. No other participants (n = 20) reported discomfort during the clinical trial. The initial electrocardiogram (performed supine and at rest before study enrolment) was normal . The vasovagal reaction occurred unexpectedly at rest directly after the start of recordings. The subject remained conscious during vasovagal reaction but showed decreased postural tone and auditory impairment. The study he was participating in was approved by the ethical committee of Zürich, Switzerland (2018-00006). Written informed consent was obtained before study enrolment and for the publication of this case report. This clinical trial is accessible on ClinicalTrials.gov (NCT03439202).The 20-year-old subject was a tall, thin  male flight attendant with no significant medical history. The subject was participating in a study investigating cerebral responses to low intensity cycling, in a small room of 22 mElectroencephalography activity signals were recorded from 19 bipolar EEG channels and sampled at 200 Hz. Pre-processing was carried out with custom-written MATLAB code (MathWorks Inc.) and the EEGLAB analysis tools . The datContinuous blood pressure was measured at the middle and index finger of the left hand using a double pneumatic cuff  and acquired at 500 Hz. Baroreflex sensitivity was calculated using the sequence method  by (1) e® 2015a . Middle cerebral artery velocity was measured in the left middle cerebral artery trough left temporal window using a transcranial doppler  and acquired at 500 Hz. Finger arterial oxygen saturation was monitored using a finger oximeter  and acquired at 0.5 Hz. Cerebral and muscular oxygenation was measured using near-infrared spectroscopy technology  and acquired at 1 Hz. Two detection and emission probes were located on the forehead and on the vastus lateralis in order to measure tissue oxygenation index (TOI), defined as the ratio of oxy-hemoglobin to change of total hemoglobin concentration, and relative concentration of total hemoglobin  in cerebral and muscular areas.Heart rate and R-R intervals were recorded using a heart rate monitor . Ectopic beats in the R-R series were compensated for by interpolation of means to obtain normal-to-normal intervals. Mean heart rate and root mean square of the successive differences (RMSSD) were computed from the normal-to-normal intervals and spectral power in the high-frequency band  was computed using the Welch method after resampling normal-to-normal intervals at 4 Hz. All signal processing was performed using MATLABThe participant remained seated for 45 min during device installation and experimental trial preparation. He demonstrated a normal health state at baseline , the participant remained unwell . His blood pressure gradually increased but to values lower than baseline  followed by a “slow” phase . These continuous recordings give insight to the succession of events before, during and following vasovagal reaction without syncope.The peaks in HR and TOI in the vastus lateralis, with associated blood pressure decrease and EEG systematic variations, suggest that the onset of the vasovagal reaction was at around 200 s after the start of the recordings. The decrease in blood pressure and HR are likely due to the parasympathetic over-activation (RMSSD and HF increase) known to trigger vasovagal reactions, which in turn decrease cardiac output . BarorefThe participant reported being anxious in the reduced environment where the measurements took place. In addition, the participant’s equipment may have added to his anxiety. The start of the recordings may have triggered the vasovagal reaction and pre-syncope.The EEG is among the first parameters to change and to recover at around 550 s, whilst blood pressure, heart rate, and MCAv remained abnormal. As a consequence, cardiac output and cerebral perfusion were reduced. In this case, cerebral hypoperfusion may be limited by the fact that the participant was seated (as opposed to standing) during the vasovagal reaction, which may explain why he did not lose consciousness. Despite limited orthostatic pressure, the parasympathetic over-activation  still induced blood pooling in the lower limbs , likely We have presented simultaneous physiological recordings during spontaneous and unexpected vasovagal reaction without loss of consciousness in a young individual. The onset of the vasovagal reaction was associated with a peak in heart rate and progressive decrease in blood pressure, with a simultaneous drop in alpha power. Parasympathetic over-activation led to hypotension and cerebral hypoperfusion. His emotional status (anxiety and feeling of oppression) and concomitant prolonged hypotension and bradycardia with EEG flattening are in favor of a vasovagal mechanism without syncope. The recovery period was associated with EEG slowing and restoration of normal EEG pattern and arousal.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by the Ethics Committee of Zurich, Switzerland (2018-00006). The patient/participant provided his written consent to participate in this study.MA, NB, GM, and DB conceived and designed the research. MA performed the experiments and drafted the manuscript. MA, NB, HM, and JB analyzed the data and prepared the figures. MA, NB, JB, GM, and DB interpreted the results of the experiments. MA, NB, EN, JB, GM, and DB edited and revised the manuscript. All authors approved the final version of manuscript.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.


Multivariable forward stepwise linear regression analysis revealed that age  and serum log-A-FABP level  were significantly associated with VRI values in KT patients. We concluded that serum fasting A-FABP level is negatively associated with VRI values and plays a role in endothelial dysfunction of KT patients.Adipocyte fatty acid-binding protein (A-FABP) is abundantly found in mature adipocytes and is involved in cardiovascular disease. Our aim is to investigate the association between serum A-FABP levels and endothelial function among kidney transplant (KT) patients. Fasting blood samples were obtained from 80 KT patients. Serum A-FABP levels were measured using a commercially available enzyme immunoassay kit. Endothelial function and vascular reactivity index (VRI) were measured using digital thermal monitoring test. In this study, VRI < 1.0, VRI 1.0–1.9, and VRI ≥ 2.0 were defined as poor, intermediate, and good vascular reactivity, respectively. There were 12 (15.0%), 30 (37.5%), and 38 (47.5%) KT patients categorized as having poor, intermediate, and good vascular reactivity, respectively. Increased serum levels of alkaline phosphatase ( The risk of cardiovascular diseases (CVDs) in patients with a functioning graft remains higher than that in the general population. Therefore, determining CV risk factors among patients after kidney transplant (KT) may lead to risk modification to improve survival . DespiteFatty acid-binding proteins (FABPs), a family of 14–15 kDa intracellular chaperones that bind to long-chain fatty acids and other lipids, were first identified in 1972 . Additiop = 0.012), γ-glutamyltranspeptidase , and serum A-FABP levels (p < 0.001) were significantly increased as VRI decreased among KT patients. Of these KT patients, 39 (48.8%) had DM and 31 (38.8%) had HTN. The most commonly prescribed immunosuppressive agents were tacrolimus , cyclosporine , mycophenolate mofetil , steroids , and rapamycin . There were no significant differences in sex, modes of transplantation, presence of DM or HTN, or the use of immunosuppression medications among the three VRI groups.Clinical characteristics and immunosuppressive drugs used in KT patients are presented in r = −0.305, p = 0.006), serum ALP level , log-transformed GGT level , and serum log-transformed A-FABP level  were negatively correlated with VRI values in KT patients. Furthermore, after adjusting with the variables that were significantly associated with VRI values, advanced age , and high serum log-transformed A-FABP level  were significantly and independently associated with VRI values in KT patients as determined by multivariable forward stepwise linear regression analysis. To better visualize the results, two-dimensional scattered plots of VRI values with age, serum ALP level, log-GGT level, and log-A-FABP level among these KT patients were drawn, which are presented as Correlations between clinical variables and serum VRI values determined by simple linear regression analysis and multivariable forward stepwise regression analysis of the 80 KT patients are presented in This study showed that advanced age and serum levels of ALP, GGT, and A-FABP were negatively associated with VRI values measured by digital thermal monitoring in KT patients. Furthermore, after adjusting with multivariable linear regression analysis, there was a significant and independent association between age or serum level of A-FABP and VRI values in KT patients.The endothelium is a target of injury during or after transplantation. Endothelial dysfunction can be measured by serum or urinary biomarkers, which can also indicate CV health and predict mortality in KT patients ,3. Over A-FABP is one of the aberrantly secreted adipokines by adipose tissues, and it is overproduced in adipose tissues in obese individuals ,18. MoreAmong all possible factors in the development of endothelial dysfunction, advanced age plays an important role . Age-assThroughout population discovery and experimental findings, recent studies proposed the role of ALP and GGT in CVD development ,30. KunuThis study has some limitations, which warrant discussion. First, this was a cross-sectional study conducted at a single center, with a limited number of KT patients. Second, our study did not measure microalbuminuria or proteinuria, although there were studies reported that serum A-FABP levels are positively associated with microalbuminuria in newly diagnosed type 2 DM patients  and micrIn conclusion, to the best of our knowledge, this is the first study to demonstrate that fasting A-FABP level was negatively associated with VRI, which is representative of endothelial function as measured by digital thermal monitoring in KT patients.In total, we examined 80 KT patients enrolled at a medical center in Hualien, Taiwan, from September 2015 to February 2016. Patients who had systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg or were receiving any anti-HTN medications in the previous 2 weeks were diagnosed with HTN. Patients were diagnosed with DM if their fasting plasma glucose level was either ≥ 126 mg/dL or if they were using oral hypoglycemic medications or insulin. Status of smoking and usage of medications were reviewed by medical records. Patients were excluded if they had an acute infection, acute myocardial infarction, heart failure, acute transplant rejection status, or malignancy at the time of blood sampling. The Protection of the Human Subjects Institutional Review Board of Tzu-Chi University and Hospital approved this study (IRB104-27-B). All patients provided written informed consent before participating in this study.Patient body weight was measured to the nearest 0.5 kg and body height to the nearest 0.5 cm, while wearing light clothing and without shoes. Body mass index (BMI) was calculated as weight in kg divided by height in meters squared. g for 10 min and then were tested for serum levels of blood urea nitrogen, creatinine, glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), calcium, phosphorus, GGT, and ALP using an auto-analyzer . The estimated glomerular filtration rate (eGFR) was calculated by the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation. Serum A-FABP and intact parathyroid hormone (iPTH) levels were measured using a commercially available enzyme immunoassay  and enzyme-linked immunosorbent assay , respectively. Fasting blood samples (approximately 5 mL) of all patients were immediately centrifuged at 3000× After overnight fasting, and resting in a supine position in an ambient temperature of 22–24 °C for 30 min, digital thermal monitoring of endothelial function was performed by utilizing a computer-based thermometry system of an FDA-approved device . Standard sphygmomanometer cuffs were placed on right upper arm of the subject. Skin temperature sensors were affixed to both index fingers of the subject . Digital thermal monitoring of both hands was obtained during 5-minute stabilization, 5-minute cuff inflation, and 5-minute deflation using an automated, operator independent protocol. The right upper arm cuff  was rapidly inflated to 50 mmHg greater than systolic blood pressure for 5 min and then rapidly deflated to invoke reactive hyperemia distally. Thermal changes during a 5-minute arm-cuff-induced reactive hyperemia test were monitored continuously in the fingertip of both the occluded  and the non-occluded  arms using VENDYS software. During the cuff occlusion, fingertip temperature in the right index finger falls because of the absence of warm circulating blood. The occlusion of blood flow elicits a vasodilatory response in the ischemic area. Once the cuff is released, blood flows into the forearm and hand, causing a temperature rebound in the fingertip of right index finger, which is directly proportional to the reactive hyperemia response. Finally, the area under the temperature curve was used to determine the VRI by taking the maximum difference between the observed temperature rebound curve and the zero-reactivity curve during the reactive hyperemia period [t test. Since the distributions of fasting glucose, triglyceride, blood urea nitrogen, creatinine, GGT, iPTH, and A-FABP levels were skewed, data were then log-transformed to achieve normality. Variables correlated with VRI values were evaluated by simple linear regression analyses; those variables significant in the simple linear regression analyses were further analyzed by a multivariable forward stepwise regression analysis. Data were analyzed by using SPSS for Windows . The value p < 0.05 was considered statistically significant. Data are expressed as means ± standard deviation (SD), and normal distribution was determined by Kolmogorov–Smirnov test. Differences among groups  were analyzed using Kruskal–Wallis analysis for parameters not normally distributed or a one-way analysis of variance (ANOVA) for normally distributed data followed by the Fisher’s protected


Hypertension often presents with microvascular rarefaction (MVR), which is closely associated with impaired angiogenesis. Early detection of MVR is essential for systemic assessment in patient with hypertension. We aimed to determine the systemic MVR through both optical coherence tomography angiography (OCTA) and intravital capillaroscopy, and to investigate their respective efficacies and related mechanisms associated with late endothelial progenitor cells (LEPCs) dysfunction.Seventy-one hypertensive and sixty-nine age-match normotensive subjects were included in this study. All subjects received intravital capillaroscopy for skin capillary density (SCD) and OCTA for retinal capillary density (RCD) and non-perfused areas (R-NPA). Subsequently, correlation of LEPCs activities and microvascular rarefaction were examined.P < 0.01] and higher R-NPA . SCD correlated positively with RCD but negatively with R-NPA . The discriminative powers of RCD performed best  followed by R-NPA  for systolic blood pressure. Similar pattern is also found for diastolic blood pressure . Furthermore, LEPCs tube formation was impaired in hypertensive patients . After multivariate adjustments, positive correlation existed between RCD or R-NPA with LEPCs tube formation  but not with SCD .Compared with normotensive subjects, hypertensive patients had significantly lower RCD [(52.9 ± 2.9)% vs. (57.8 ± 1.6)%, Compared to intravital capillaroscopy, OCTA is a more precise technique for early detection of hypertensive microvascular rarefaction, which is associated with the fall in LEPC-mediated angiogenesis. Both of OCTA and LEPCs function can help identify hypertension-related capillary abnormality.Trail Registration The trial is a substudy of EXCAVATION-CHN1, registered at clinicaltrials.gov as NCT02817204 . Hypertension is one of major contributing factors to the development of atherosclerotic cardiovascular diseases (ASCVD) . Recent Recently, optical coherence tomography angiography (OCTA) gradually emerges as a new technique for the evaluation of retinal microcirculation in clinical practice , 13. WitEPCs is a specialized subset of hematopoietic cells found in the adult bone marrow and peripheral circulation arising from hemangioblasts prenatally. EPCs are substantially crucial for the preservation of a structurally and functionally intact endothelium and the maintenance of the normal endothelial integrity , 16. EPCIn light of these, we hypothesized that OCTA can identify hypertensive MVR in an early phase, as observed by conventional capillaroscopy, and that microvascular abnormality may be correlated with LEPCs dysfunction, as suggested by previous observations. Also, we would like to find out which method of detection is superior for early detection of hypertensive microvascular rarefaction. Therefore, we enrolled hypertensive patients and age-matched normotensive volunteers. First, we investigated whether OCTA can effectively detect MVR in mild hypertension; second, the correlation between OCTA and traditional intravital capillaroscopy, and the association of retinal microvascular perfusion and blood pressure were compared; last, LEPCs function were tested and the association between LEPC-mediated angiogenesis and MVR was examined.Microvascular rarefaction plays a key role in hypertension-induced microvascular organ damage which is associated with high morbidity and mortality. Dysfunction of endothelial progenitor cells (EPCs) leads to impaired angiogenic capacity in hypertension contributing to capillary rarefaction. In order to identify whether aerobic exercise can improve angiogenesis capacity of EPCs which contributes to ameliorate microvascular rarefaction in hypertension, EXCAVATION-CHN1 was designed and registered as NCT02817204, which was a single-center, open-label, controlled, parallel group study conducted in Guangzhou, Guangdong, China. Previous to perform exercise intervention, it is necessary to identify the status of the microvasculature and EPCs functions between normotension and hypertension.This study is the sub-study of RCT EXCAVATION-CHN1. Seventy-one essential hypertensive patients without any treatment for high blood pressure and sixty-nine normotensive subjects matched for age were enrolled, based on the inclusion criteria of EXCAVATION-CHN1: aged 25 to 40 years; blood pressure raised with home blood pressure (HBP) 140 mmHg to 159 mmHg and/or diastolic blood pressure 90 mmHg to 100 mmHg; non-smoker; no previous exposure to anti-hypertensive medication, herbal supplements, anti-depressants or other traditional Chinese medication. In addition, the rationale for exclusive male enrollment was based on the consideration that menstruation would affect the completion of the continuous exercise training protocol (5 times a week and lasts for 12 weeks) in female subjects. Other key exclusion criteria included secondary hypertension; history or presence of cardiovascular disease; diabetes or pre-diabetes mellitus or systemic immune diseases.The study consisted of two phases: first, a 2-week run-in period through HBP monitoring and baseline blood biochemical examination; second, 1-week intravital capillaroscopy and OCTA examination.BP measurement was performed in accordance with the method recommended by clinical guidelines , 19. HBP2 per field) centered on an ink spot at each site were recorded continuously for 5 min to detect intermittently perfused capillaries, images and videos were coded and stored with Videocap 8.14 software . Capillary density in the resting state was counted during a 15-second period, during which visible perfused capillaries (continuously and intermittently) were counted. We applied venous congestion (VC), with the digital cuff  inflated to 60 mmHg for 2 min, to expose a maximal number of non-perfused capillaries. Using the same visual fields that were used during resting state, the capillaries were counted in the 2-min recordings.The intravital capillaroscopy was performed with final 136× magnification on 19-inch display . The skin of the dorsum of the middle phalanx of the left hand was examined. Four microscopic fields (0.66 mmSkin capillary density (SCD) was counted offline to determine whether a capillary was present or not by a single-experienced investigator who was blinded to the group assignments from a freeze-framed reproduction of the videotape and from the running videotape, as well as the hypertensive status and blood pressure data of the study subjects.OCTA was performed using OptovueAngioVue system . After the subject was seated, the operator used joystick to move optic head with the chinrest up and down. Adjusted the mark in the IR live video on the monitor to center subject’s pupil of oculus dexter (OD) or oculus sinister (OS), moved forward slowly and asked subject to focus on the blue fixation target inside the machine, then stopped the machine until the retinal IR image reached sharpness, and executed the auto adjustment. When the adjustment finished, the operator started scanning by using a light source centered on 840 nm and bandwidth of 50 nm in order to a high-quality OCT images on the monitor. With split-spectrum amplitude-decorrelation angiography (SSADA) technique , each OCRCD and R-NPA measurements were performed on 3 mm × 3 mm retinal parafovea scan field size and were used to assess ocular perfusion impairment in vascular conditions. RCD was a measurement of the proportion of pixels occupied by flowing vessels out of the total pixels within the area of analysis (Software Version 2015.1.0). The outputs of RCD analysis included color-coded vessel map and regional flow density map. R-NPA was obtained by the software, while the outputs were the non-perfused areas by mouse click selection. Ischemic areas would be shown in yellow and be saved and matched with others in the study . OD and Late EPCs were cultured and characterized as in our previous study . Briefly4 EPCs, resuspended in 250 µl EBM-2, were pipetted in the upper chamber of a modified Boyden chamber . The chamber was placed in a 24-well culture dish containing 500 µl EBM-2 supplemented with either PBS, or 100 ng/ml SDF-1 . After 24-h incubation at 37 °C, transmigrated cells were counted by independent investigators blinded to treatment groups.LEPCs migration was determined using a modified Boyden chamber. Briefly, 2 × 105 cells in each well of a 4-well plate. HUVECs were pretreated with or without 1 ng/ml tumor necrosis factor-α  for 12 h. Then 1 × 105 CM-DiI -labeled EPCs were added to each well and incubated for 3 h at 37 °C. Nonattached cells were gently removed with PBS, and adherent EPCs were fixed with 4% paraformaldehyde and counted by independent investigators blinded to treatment groups.A monolayer of human umbilical vein endothelial Cells (HUVECs) was prepared 48 h before the assay by plating 2 × 104) were resuspended with EBM-2, and loaded on the top of the Matrigel. Each conditional group contained 3 wells. Following incubation at 37 °C for 2 h, each well was imaged directly under a microscope, and an average of tubules was counted from 3 to 5 random fields.LEPCs tube formation experiment was conducted as follows: a growth factor-reduced Matrigel (Corning) was warmed up at 4 °C overnight. After completely thawed, 60 μl of Matrigel was plated to 96-well plates at the same level to distribute evenly, and incubated for 1 h at 37 °C. LEPCs . Correlations between variables (SCD and RCD or R-NPA) were analyzed by linear regression analysis. With adjustment for confounding factors , since the dependent variables are continuous variables, and the independent variables basically conform to the LINE criteria , we adopted the multivariant linear regression model with beta value and 95% confidence interval (CI) to determine which factors were the independent predictors of LEPCs tube formation ability.The statistical analysis was performed by an independent medical statistician. Continuous variables were described as the mean ± SD for normally distributed data . Comparisons between groups were performed with Student P < 0.05. Analysis was performed using SPSS software  and figures were plotted using Prism software (version 6.0).In our study, SBP and DBP data is normally distributed. The SBP (132 ± 6.2) mmHg and DBP (80 ± 5.6) mmHg were divided into binary variable, and the classification of SBP and DBP by SCD, RCD and R-NPA was evaluated by receiver operating characteristic curve (ROC) method, using the area under the curve (AUC) as a criterion for classification. All tests were 2-tailed, and a statistical significance was established at 2 and the proportion of obese individuals (BMI 30 kg/m2). No statistically significant differences between groups were found in the main demographic characteristics including serum glucose level, dietary salt intake (obtained by self-monitoring continuously for 5 days), serum lipid level and renal function. However, BP level between normotensive and hypertensive group was significant differences   /mm2, P > 0.05). RCD was decreased  and R-NPA was increased in hypertensive patients  (Table SCD of hypertensive patients was lower than that of normotensive subjects in the resting state and after 2-min VC (both 05) Fig. A, B; add. 57.6 ± .6%, P < P < 0.01); ] , as well as between SCD and R-NPA (mean of OD and OS) because SCD by intravital capillaroscopy was the most classic and recognized approach. These data showed that SCD had moderate-intensity positive correlation with RCD and negative correlation with R-NPA [ followed by R-NPA  in SBP analysis. Similar pattern is also found in DBP analysis   Fig. Bb.P < 0.001), adhesion (normotension: (46 ± 6.6)/hp vs. hypertension: (27 ± 4.9)/hp, P < 0.001) and tube formation abilities (normotension: (37 ± 2.5)/mm2 vs. hypertension: (31 ± 4.6)/mm2, P < 0.001) were significantly declined in the hypertensive group /hp vs. hypertension: 33 ± 5.6)/hp, 3 ± 5.6/hP < 0.05), LEPCs tube number and RCD , LEPCs tube number and R-NPA   but not with SCD   both NVC and OCTA can detect MVR in early hypertension. (2) OCTA results were independently related to LEPC-mediated angiogenesis and correlated better with hypertensive blood pressure level than intravital capillaroscopy. (3) In vitro functions of LEPCs were impaired and closely correlated to MVR in hypertension. To the best of our knowledge, our current study for the first time demonstrated that OCTA and intravital capillaroscopy can be both used as effective approaches to investigate MVR in mild hypertension, indicating that combination of two method would be a better way for systemic MVR detection and OCTA might be better than intravital capillaroscopy in mild hypertension. Moreover, systemic MVR in early hypertension was correlated with in vitro LEPCs dysfunctions.MVR is an important pathophysiological process in hypertension which increases integral peripheral vascular resistance, and eventually contributes to the development of ASCVD , 22. ThuSince the SSADA technique was proposed for better refined images with a diameter less than 10 microns, quantitative numerical data and non-interfering real-time flow density map , 30, newIn this study, variables as RCD and R-NPA were first synchronously applied in hypertensive patients and showed a decrease in RCD and an increase in R-NPA. With this observation, we found not only retinal capillary structure but also blood flow was impaired in hypertension. In general, we demonstrated for the first time that structural and functional MVR were simultaneously present in retinal capillary in the early stage of hypertension. To determine its consistence, we assessed the relationship between OCTA and intravital capillaroscopy, and found both RCD and R-NPA had positive linear correlation with SCD. Our data clearly manifested the coherent trend of MVR presented in retina and skin, and documented that MVR is an integrated change in the early stage of hypertension. Previous studies revealed that chronic hypertension (more than 10 years) affects the retina OCTA parameters  and reduAlthough there are many factors involved in the development of hypertensive MVR, such as a lack in tissue VEGF, an extracellular matrix more resistant to peptidases, a defect in VEGF receptors, an imbalance between Ang1 and Ang2, and so on, accumulating data demonstrated that EPC-related deficient angiogenesis contributes to MVR in hypertension. Previous experiments confirmed EPCs dysfunction presented in SHR with MVR . Our teaMicrovascular change can be affected by age, BMI, BP, dietary salt intake, glycolipid metabolism, systemic inflammatory response and so on , 39. TheCapillary rarefaction was observed in hypertensive patients with intravital capillaroscopy, compared to normotensive individuals regardless of blood pressure levels . StudiesThe current study had several limitations. First, this is a small sample size study. A study of a larger population should be performed to validate these results. Second, patients with various levels of blood pressure should be enrolled for further exploration regarding the relationship between MVR and blood pressure levels in the future. Third, since only male patients were enrolled in this study, these results should also be replicated in female population.In conclusion, our present study demonstrates for the first time that OCTA is a more precise technique for early detection of microvascular rarefaction in hypertension compared with intravital capillaroscopy, which was associated with the fall in LEPC-mediated angiogenesis. Both of OCTA and LEPCs function can help to identify hypertension-related capillary abnormality.


Despite improvement in short-term outcome of kidney transplants, the long-term survival of kidney transplants has not changed over past decades. Kidney biopsy is the gold standard of transplant pathology but it’s invasive. Quantification of transplant blood flow could provide a novel non-invasive method to evaluate transplant pathology. The aim of this retrospective cross-sectional pilot study was to evaluate positron emission tomography (PET) as a method to measure kidney transplant perfusion and find out if there is correlation between transplant perfusion and histopathology.15 O]H2O PET. Perfusion and Doppler resistance index (RI) of transplants were compared with histology of one-year protocol transplant biopsy.Renal cortical perfusion of 19 kidney transplantation patients  and 10 healthy controls were studied by  and the group of no fibrotic changes [0.66 (0.61–0.72)] differed statistically significantly (p = 0.03). No statistically significant correlation was found between cortical perfusion and fibrosis of transplants (p = 0.56).Renal cortical perfusion of healthy control subjects and transplant patients were 2.7 (2.4–4.0) ml min15 O]H2O PET showed its capability as a method in measuring perfusion of kidney transplants. RVR of transplant patients with stage 2–3 chronic kidney disease was higher than that of the healthy, although kidney perfusion values didn’t differ between the groups. Doppler based RI correlated with perfusion and fibrosis of transplants.[ Renal allograft survival has improved over past decades mainly because of increased first-year survival , 2. HoweChanges in renal tissue oxygenation and microvasculature are considered as major determinants of progression of chronic kidney disease (CKD), irrespective of its cause , 6. MicrKidney biopsy, which is the gold standard of evaluation of transplant parenchyma, is invasive and prone to sampling errors . On the Quantification of renal blood flow (RBF) has been challenging because of complicated vascular structure of the kidneys as well as difficulties in finding quantitative and noninvasive measuring techniques. Several semi-quantitative methods have been used to evaluate renal allograft perfusion like scintigraphy , dynamicDoppler-echo with resistance index (RI) is based on blood velocity and blood pressure, thus it measures perfusion indirectly. RI has been used to monitor allograft function at predetermined time points after transplantation as well as in acute settings. However, conflicting data has been reported of its value in reflecting allograft condition and histology –22.15O]H2O positron emission tomography (PET) is a non-invasive and quantitative method to measure regional one-kidney perfusion without contrast agent [15O]H2O PET and compare transplant perfusion to transplant biopsy and Doppler RI.H2O. A low dose helical CT scan with automatic dose modulation  was acquired during normal breathing before the PET scan to correct for photon scatter and attenuation. Thereafter 700 MBq of [15O]H2O was given and PET scanning was conducted. Blood pressure and heart rate (HR) were measured by an automatic oscillometric blood pressure machine.The subjects were positioned supine in the camera [Discovery 690 PET/computed tomography (CT)] scanner, GE Medical Systems, Waukesha, Wisconsin, USA). Venous catheter was placed in an antecubital vein for injection of H2O into and from tissue, Va is the vascular volume fraction, and delay parameter accounts for the difference of radioactivity appearance times between the blood and tissue curves. K2 multiplied by physiological partition coefficient (pphys) is renal perfusion (ml min− 1 g− 1) . RVR tended to be higher in the group of mild inflammatory changes than in the group of no changes .There were 9 biopsies in the group of no inflammatory changes (7 biopsies with score 0 and 2 biopsies with score 1), and 8 biopsies in the group of mild inflammatory changes . No statistically significant difference was found in eGFR, renal perfusion, RVR, 24 h MAP or Doppler RI between the groups , RVR of the patients was statistically significantly higher than that of the healthy. Furthermore, Doppler RI of transplants correlated with transplant perfusion and fibrosis. However, somewhat surprisingly, there was no correlation between transplant fibrosis and perfusion.− 1 g− 1 being similar with other studies by [15O]H2O PET, in which renal cortical perfusion in the healthy has varied between 1.6–4.7 ml min− 1 g− 1 [15O]H2O PET based renal perfusion studies lower kidney perfusion has been demonstrated in patients with CKD than in the healthy [− 1 g− 1 of transplanted kidneys and that of the healthy controls. However, CKD stage was more advanced in the patients of previous studies than in the patients of our study probably explaining the difference.Renal cortical perfusion in the healthy was 2.7 (2.4–4.0) ml min− 1 g− 1 , 30, 40. healthy , 27. In RVR describes the resistance to blood flow offered by renal blood vessels. Although renal perfusion values between the groups were the same RVR was higher in transplant patients than in healthy controls probably reflecting microvascular dysfunction in the kidneys of transplant patients. Because systolic blood pressure and MAP were statistically significantly higher in transplant patients than in controls kidney perfusion values didn’t differ between the groups. In other words, increased blood pressure maintained renal perfusion in transplant patients.Hetzel et al. demonstrated similarly an increased RVR in transplant patients compared to controls by PAH (para-aminohippurate) – technique . Also inThere are several reasons for microvascular dysfunction in our study. Especially, calcineurin inhibitors are associated with increased vascular resistance , 43. PerOur initial hypothesis was, that the decrease in perfusion would correlate to the changes in kidney transplant histopathology. However, we could not verify any correlation between transplant perfusion or RVR and fibrosis grade of kidney biopsy. Inflammatory changes in kidney biopsy and RVR tended to correlate.In some Doppler based studies an inverse correlation between transplant perfusion and fibrosis has been shown , 32. HowTransplant RI and perfusion correlated inversely in our study. Similar correlation has been shown early after transplantation . TransplFibrosis of transplants seemed to increase in parallel with increasing Doppler RI. Other studies have demonstrated highly variable results concerning transplant histology and RI. Like in our study, Radermacher et al. showed a correlation between transplant fibrosis and transplant RI . Gao et There was a statistically significant correlation between eGFR and perfusion and RVR in the healthy but not in the patients with kidney transplant. One reason for this discrepancy might be variable perfusion contribution from native kidneys in transplant patients although the effect of native kidneys on perfusion is known to decrease after transplantation .There are some limitations in our study. Some transplant biopsies were not representative according to Banff criteria, especially arteries were partly lacking. However, the number of vessels should not have influenced the fibrotic and inflammatory scores that we were interested in. In addition, there was quite a long period between PET-imaging and kidney biopsy which may have influenced on our results. However, the average kidney function of patients was only mildly decreased during that time likely indicating that kidney biopsy histology would not have changed remarkably either. Furthermore, it is likely that cortical ROIs included an unknown admixture of medullary flow due to partial-volume effect and spatial resolution thus decreasing perfusion values. However, that phenomenon was similar both in the healthy and in the patients not influencing the difference in perfusion values between the groups. In addition, the state of autonomic nervous system which is known to influence renal perfusion  may haveIn conclusion, this pilot study showed the capability of PET-technique to measure kidney transplant perfusion. RVR of patients with kidney transplant was increased compared to healthy controls reflecting microvascular dysfunction of transplants. Further studies on larger number of transplant patients with advanced stage of CKD and varying degrees of fibrosis are needed to reveal the possible correlation between transplant fibrosis and perfusion.Additional file 1: Figure S1. Aortic ROI of the healthy control subject.Additional file 2: Figure S2. Iliac ROI of patient with kidney transplant.Additional file 3: Figure S3. Image-derived arterial blood TACs from a control subject and a kidney transplant subject.Additional file 4: Figure S4. Iliac artery ROIs in a three-dimensional PET image.


Hypertension is a major risk factor related to leading causes of death among older adults. Numerous efforts have been done but they still remain sub-optimal. This condition encourages development of a non-pharmacological therapy to complement pharmacological therapy, such as progressive muscle relaxation and music therapy (RESIK). The purpose of this study was to determine the effect of RESIK on blood pressure among older adults with hypertension in Depok Indonesia.This study used quasi experimental design with pre and post test with control group approach. One-hundred older adults with hypertension were divided into two groups using stratified random sampling and purposive sampling.After 11 sessions of RESIK therapy in 6 days, the paired t-test showed a decrease in systolic blood pressure to 29.2 mmHg and a decrease in diastolic blood pressure to 16.2 mmHg.p < 0,001, but it did not significantly decrease diastolic blood pressure p > 0.167. It is recommended that RESIK be used regularly for an older adult population with hypertension.In conclusion, RESIK decreased systolic blood pressure  Hypertension is one of the leading causes of death, worldwide. As many as 9.4 million people die every year due to hypertension, and more than 1 billion people live with high blood pressure, 40% of which are over the age of 25 . MoreoveThe Indonesian government has undertaken efforts to surveillance of Non-Communicable Diseases (NCDs) based Posbindu and pharmacological therapy . HoweverNon-pharmacologic therapy, such as progressive muscle relaxation and music therapy (RESIK), is a type of nursing intervention used to decrease blood pressure . Many stThis research used music therapy in conjunction with progressive muscle relaxation. Music therapy was a therapy that could help in the healing process. Music that could be used in this therapy was a consistent and stable rhythm, dynamic, fun harmonious, regular rhythm without any sudden change . The insThis study used quasi experimental design with pre and post test with control group approach. In the present study, the sample consisted of 100 older adults with hypertension in Depok, Indonesia. The participants were divided into an intervention group and a control group. The sample size in the study was determined using different formula 2 mean dependent group that had considered the drop out sample and the design effect. Sampling method used stratified random sampling. Researcher conducted two times drawing, the first drawing was to determine the research area, from 11 districts in Depok City, researcher got Cimanggis as a place for research, followed by the second drawing which aimed to determine the district used as the intervention group as well as the control group. Results from the second drawing, from 6 Sub-districts in Cimanggis, Harjamukti Sub-district was obtained as research area for intervention group and Cisalak Pasar for control group, next the researcher used purposive sampling to choose community groups which were used as research place. The inclusion criteria were: older adults with systolic blood pressure ≥ 140 mmHg and diastolic ≥90 mmHg, with no hearing and vision impairment, no mobilisation impairment or cognitive disorder, with a mini-mental state exam (MMSE) score ranging from 18 to 23 and who had or had not undergone standard anti-hypertension therapy. The exclusion criterion was older adults with critical hypertension, characteris ed. by systolic blood pressure ≥ 180 mmHg or diastolic blood pressure ≥ 120 mmHg.Sampling procedure can be seen in Fig. The study was conducted as many as 11 sessions in 6 days in the morning and evening with 3 practice sessions. The exercise schedule took one week to get maximum result –15. Six The present study has received ethical clearance and was approved by the Faculty of Nursing Ethical Committee of the Universitas Indonesia on 17 April 2017. The intervention caused no harmful side effects for the respondent if the respondent did in accordance with the procedure, but if the respondent did not follow the procedure accordingly, blood pressure would increase. If the respondents’ blood pressure increased during therapy, they were encouraged to rest while breathing regularly for 5 min until they felt relaxed.The mean age of the respondents in the intervention group was 66.54 (standard deviation [SD] = 5.665). In the control group, the mean age of the respondents was 67.48 (SD = 5.388) with a homogeneity score of 0.397. All the characteristic variables were tested for homogeneity using a chi-square test for the categorical data and an independent t-test for the numerical data. The results showed that the respondents characteristics in the intervention and control groups were homogeneous. Other respondent characteristics are presented in Table Participating in progresive muscle relaxation until feeling relaxed, calm and while fully concentrating for 30 min can decrease a person’s corticotrophin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) levels in the hypothalamus . This prThis study showed that blood pressure decreased as many as 29.2 mmHg (systolic) and 16.2 mmHg (diastolic), the detailed results are scribed in Table The research suggested that music could inhibit and balance brain waves, capable to activate limbic system related with emotion. When the limbic system was activated, the individual would feel relaxed. The music could affect sympathoadrenergic activities that had a role in plasma catecholamine concentrations and also affected the release of stress-released hormones and stimulated the body to produce nitric oxide (NO) molecules that working on blood vessel tone and could decrease blood pressure –21.Changes in blood pressure are related to the administration of anti-hypertensive drugs, which are routinely used by older adults. Research has shown that captopril can decrease systolic blood pressure and diastolic blood pressure by as much as 29.16/11.83 mmHg and amlodipine can decrease systolic blood pressure and diastolic blood pressure by as much as 32.94/16.38 mmHg . Based oTable  shows noOther factors that may affect diastolic blood pressure include tea drinking habits, triglyceride levels, lipoproteins, blood glucose and Body Mass Index (BMI) . This fiIn the present study, blood pressure was sometimes increased after treatment. This outcome occurred in 5 respondents because 4 of them did not follow the protocol, such as exhaling through the mouth, and they remained tense when the researchers instructed them to relax their muscles and 1 respondent felt worried about her blood pressure. The intervention protocol indicates that while respondents are attempting to relax, nurses or researchers must consider how to relax the muscle group a respondent is tensing. If respondents do not achieve a relaxed state, CRH is secreted and ACTH levels in the hypothalamus are not optimal, so the parasympathetic nervous system releases the neurotransmitter acetylcholine to inhibit the sympathetic nerves by reducing the contractile heart muscle; moreover, vasodilation of the anterior and vein was also not optimal so the blood pressure could not significantly decrease .RESIK therapy has to be done regularly to maintain blood pressure within normal limits. RESIK in this study was a coping mechanism regulator that could affect physiological function, self concept, role function and interdependensi elderly. These effects would provide both adaptive and ineffective responses . Roy’s tEvidently, the mean of systolic and diastolic blood pressure in the intervention group included in the pre-hypertension category. That was different with control group. The mean of blood pressure in the control group was in category 1 hypertension. While diastolic blood pressure was no difference significantly with the control group, this therapy could help maximizing the treatment that has been performed such as drug administration as previously explained that the decrease in blood pressure in the intervention group was higher than the control group who had the habit of taking medicine as many as 74%.In the present study, RESIK therapy significantly decreased blood pressure, dropping it from category 1 hypertension to pre-hypertension. RESIK therapy should be administered in accordance with the implementation protocol to obtain an optimal therapeutic effect.


Purpose: Increasing attention is being paid to the role of the intelligent self-management of hypertension under the context of increasing prevalence but limited medical resources. However, heterogeneity in interventions and outcome measures has hindered the interpretation of research evaluating mobile health technologies for hypertension control, and little study of such technology has been performed in China. Objective: This was a feasibility study aimed to understand patient and medical practitioners’ acceptance and experience of a mobile-phone based platform for the management of hypertensive patients. Methods: The model used behavioral incentives for daily blood pressure measurement and physician-facing prioritization of patients based on level of blood-pressure control. Patients were enrolled by purposive sampling. The platform was used for two-week blood pressure monitoring through WeChat, which simulated our future app. Qualitative interviews with patients and providers were conducted in time. Results: Twenty hypertensive patients and two providers were enrolled and used the platform throughout the two weeks. Patients reported daily home blood pressure monitoring to be simple, feasible and increased their health awareness. Specifically, patients self-reported that reminders, the daily frequency and time of monitoring, and positive reinforcement were important for maintaining adherence. Providers reported that they could manage patients more quickly and accurately, but reasonable feedback information was needed to avoid excessive increases in workload. Conclusion: The adoption of mobile-based technology to monitor patient’s blood pressure may provide a practical solution for managing patients in Chongqing, China. Patient health education and enhanced app functionality could improve patient compliance and satisfaction while reducing provider workload. Hypertension, or elevated blood pressure, is a major risk factor for cardiovascular disease and related deaths  though pWe envisaged designing and testing a home-based self-management model for hypertensive patients in Chongqing, China, aiming to improve the efficiency in blood pressure control under the context of limited medical resources. Patients would complete their blood pressure measurements at home every day and the blood pressure values could be inputted into an application automatically thorough Bluetooth instead of entered by patients manually. WeChat is a social app commonly used in China, which is easy to operate and extremely popular among middle-aged and elderly people. According to official data released by WeChat, as of September 2018, there are 1082.5 million active users every month, and 63 million active users are over 55 years old . We condBefore this study, two tests (beta test one and beta test two) had already been completed in four living labs . In beta test one, ten non-patient volunteers were recruited per lab and we gathered user experience on daily measurements as well as the reminder and behavior feedback messages. In beta test two, two or three care providers per living lab were interviewed to identify essential features of the provider’s dashboard and evaluate the proposed user flow .In this study, we monitored patients’ blood pressure regularly using WeChat to simulate our future app and provided feedback to health care providers using a dashboard designed by experts. After two weeks of monitoring, we interviewed patients and providers who participated in this process. This study was approved by the Ethical Committee of Chongqing Medical University .Considering the operability of this feasibility study, we selected two districts close to Chongqing Medical University, which, at this stage, were relatively easier to coordinate and connect with our research sites. Two communities in Jiulongpo District and Nan’an District were purposively selected as our research sites. A sample of ten hypertensive patients was recruited by the provider at each site using the method of purposive sampling to ensure that our sample reflected a gender balance and a wide range of ages. Inclusion criteria were the diagnosis of hypertension without severe cognitive impairment and ability to use WeChat with a smart phone. Patients were informed of the study risks and included if they signed the informed consent. Subsequently, patients were provided, enrolled, and received training on home use of the electronic sphygmomanometer (OMRON HEM-7120). The participants’ basic characteristics, which were collected during the training session, included blood pressure at entry, treatment plan, and frequency of measuring blood pressure prior to joining the study. Based on the National Institute for Health and Care Excellence (NICE) guidelines , a fourtIn the thirty minutes prior to obtaining a blood pressure measurement, patients are required not to do any strenuous exercise, drink coffee. or smoke. If possible, patients also need to urinate before measurements. Patients are supposed to rest for at least five minutes before the measurement. During the measurement, patients should sit at the table with their feet flat on the floor and their back against the back of the chair, then put arms at the heart level and relax them on the table. Patients should take off any clothes that are too tightly bound to the upper arm, and preferably take a measurement from the left arm. If the blood pressure measured in the right arm is higher or patients are not allowed to use the left arm for medical reasons, the blood pressure can be measured in the right arm. Patients are required to make sure that blood pressure is measured on the same arm each time. They should put the cuff on the left arm, the lower edge of the cuff should be three centimeters above the elbow bend, the instrument wire needs to be in a visual position and point to the middle finger, and the arm should tighten slightly—not too tightly or too loosely. Patients cannot clench their fists during the measurement. When the measurement is completed, patients need to record their values (systolic blood pressure and diastolic blood pressure) displayed on the instrument. They should measure their blood pressure twice again in the same way, with an interval of one minute, and record the values.After obtaining their measurements, the results are sent to the research assistant. A double check was completed by two researchers together when blood pressure values were received and before the average values were sent to care providers. We handled false measures in two ways: if the three values of the day were obviously different, we would ask the patient to re-measure the blood pressure three times as soon as possible and record the results; or if the average value of the day was abnormal compared with the past few days, it would be handled by the care providers.The two-week feasibility study was mainly carried out by a trained postgraduate medical student. A WeChat message to remind patients to measure their blood pressure was sent at a fixed time (nine o’clock) every morning. If patients did not provide feedback on their blood pressure after one hour, they would be reminded again (only once per day). The student collected the self-reported blood pressure values of patients  from the two sites every day, calculated the average value of each patient and then prioritized average values according to a set of pre-defined criteria. The four categories conformed with the Chinese national hypertension and treatment guidelines : Red—theAfter the two-week home blood pressure monitoring through WeChat, twenty patients and two providers were interviewed. The interviews were conducted in the offices of the two sites through individual in-depth interviews and were presided over by the professional investigators of the research team, with an observer and recorder, respectively. Before the interview, the interviewers were trained to ensure the interview process could be conducted according to a unified standard. Interviews were recorded after consent to ensure the comprehensiveness and reliability of the results.A special group interview was organized at the two research sites, and each panel was organized by two researchers. One was responsible for questioning and guidance, and the other was responsible for observation and documentation. Participants, under the guidance of the organizers, were asked the same questions and expressed their opinions on different issues in turn. The focus group discussions (FGDs) were recorded with the consent of the participants, and each group discussion lasted approximately 30–40 min. The interview of patients was semi-structured. After discussion by experts, four key themes were addressed based on the “Guidelines for the Prevention and Treatment of Hypertension in China (2018 Revision)”  and betaAfter the interview, the recordings were transcribed verbatim by the local postgraduate medical students. Similar results were marked and a thematic framework for analysis and summary was established based on this. The transcribed results were encoded using Maxqda11 qualitative analysis software.P < 0.05) in systolic blood pressure between the values we recorded when they were enrolled versus after one-week of monitoring  said it took less than five minutes, and none required more than ten minutes. “In less than five minutes. After the machine was tied to my hand all I needed to do was press it. Maybe it just (took me) two minutes.”“It took (me) less than ten minutes to measure my blood pressure three times and average the values.”In addition, all patients thought the process of measurement was simple and the instrument was easy to operate, but two of them reported the process of measurement was a bit boring for them, even though the operation was not difficult.“It’s easy enough (for me to complete the measurement).”“It’s easy to use the instrument. I just think the less trouble, the better. The process is a bit boring for me.”“Every time I received the reminder, I felt grateful.”“I am impressed with the message, (referring to the information on the phone), ‘You measured your blood pressure very positively yesterday, please stick with it tomorrow.’”“It had me insisting to take the measurement. I had forgotten to measure my blood pressure at the set time somedays, and felt even a little guilty when I received the reminder again one hour later.”Although the students used WeChat to remind patients to monitor their blood pressure every day during the study, some patients (7/20) indicated they did not recall the precise content of messages they received . Patients who remembered the details showed a positive attitude toward the reminders:All the patients expressed satisfaction with the length and tone of the messages they received. Four patients reported that it would be nicer if the messages could be diversified.“Well, I felt pretty satisfied. I think it was a concern for the elderly. The message wasn’t too long. No matter what you sent me, it was a motivation to us, which wasn’t annoying.”Fifteen of the twenty patients indicated that the two-week home blood pressure monitoring made them feel healthier. The other five patients did not report an impact on their self-perceived health—among them, four believed it was not necessary to measure blood pressure every day, as s/he felt that s/he would be okay if they just took their medicine, and one patient did not feel a change in health because he already had the habit of daily blood pressure monitoring. “I was in a better mood (with the measurement during the study), I also recorded my own blood pressure every day. I was really satisfied that my blood pressure was quite normal at this age.”“At the very least, I knew more about my own blood pressure.”“It had little effect on me. In fact, I used to measure blood pressure myself (before this study).”Based on records from the dashboard during the two-week study interval, half of the twenty patients (10/20) performed daily measurements, six patients measured thirteen times, one patient measured eleven times, two patients measured ten times, and only one patient measured seven times during the study. When patients were asked about the reasons why they measured their blood pressure, the vast majority (19/20) said it was because they believed it was good for their health. Seven of them said they enjoyed the process, which was also one of the reasons why they performed the measurements. One patient indicated that measuring or not measuring their blood pressure was equivocal.“It’s not entirely because of the benefits, I also like the measurement.”“Actually, I don’t think it is necessary to measure the blood pressure. I never felt uncomfortable even when my blood pressure was 160 or 170.”Most patients (17/20) believed it would be easier to stick to measuring at a flexible time every day rather than a set time.“I prefer to measure my blood pressure at six o’clock am every day, I usually begin to exercise at 6:30, so I have to take the sphygmomanometer with me every morning to ensure my blood pressure value could be fed back at the specified time these days.”“I am more willing to measure my blood pressure from ten to eleven o’clock am, because I need to buy food earlier than this period, and then prepare lunch for my grandchildren.”“I would like to measure earlier in the morning if I need to go out in the afternoon, but I prefer to measure my blood pressure in the afternoon if I am busy doing housework in the morning.”When asked about their views on daily vs. weekly blood pressure measurements over the two-week period, half of the patients (10/20) believed daily blood pressure measurements were better, while the other eight patients felt daily measurements were not necessary. Two patients were neutral and thought weekly or daily blood pressure measurements were both acceptable.“I think it’s better to measure blood pressure every day. The value can be different after two minutes.”“In fact, I think it (blood pressure) should be measured every day. Since you gave me this instrument I have measured my blood pressure twice a day.”In the interview, we also asked patients for opinions about using a related app to monitor blood pressure in the future rather than using WeChat. None of them (0/20) thought using WeChat every day was a great challenge to them, but eight of them thought using an app which could provide feedback on blood pressure automatically without inputting the blood pressure manually would be better than WeChat. Another twelve patients felt using either WeChat or the app was feasible for them. “It will be more labor-saving to receive feedback automatically by an application, and it’ll be nice if we do not need to input our blood pressure ourselves.”“Both (application or WeChat) works for me. Although I could not input my blood pressure values manually by typing, I could use voice to feed back the values through WeChat.”Most patients (14/20) clearly indicated their willingness to measure their blood pressure in the future (daily or weekly). They believed they developed a habit during the two-week intervention. Two patients indicated either daily or weekly measurement in the future were both acceptable for them.“I’d like to (measure blood pressure in the future), since I have developed the habit during these days.”“Sure, I think it’s a good thing and a responsibility for our health.”Providers were in agreement across the two sites and they checked the dashboard daily and provided usual care to the patients based on the standardized guideline which was provided during training. After receiving the categorized and prioritized dashboard with patients’ information, they were most concerned about patients in the red and orange categories. Additionally, mean values and fluctuations of blood pressure were used as references for the treatment of patients.“Patients in the orange category were those whose blood pressure was not well controlled, so I mainly paid attention to this area. There were also a few patients in the red category. I tended to use the fluctuation of the blood pressure over one week  as the b“I started with the red category and especially cared about the details of those unusual values; I would look at their mean values and fluctuations of blood pressure, so that I could determine whether the high value was caused by improper measurement methods or exercise prior to measurement.”“If patients appeared in the red category, they would be called into the clinic for the treatment; otherwise, patients would be observed for another period of time, and would be notified to come into the clinic if the value was still abnormal.”“The management model of daily monitoring increased the communication time between the patients and I, particularly for patients with uncontrolled blood pressure. It would be quicker (to check the dashboard) if their blood pressure was well controlled.”“It would be better if a weekly average of patients in the orange category and daily high-risk situations (red category) of patients were reported in the dashboard, and I preferred patients’ blood pressure values be ranked from high to low.”Both providers believed the dashboard was extremely concise and clear and could help them understand their patients’ blood pressure better. However, there were also some concerns:“During this study, we did not provide feedback to patients in the green category (those with controlled hypertension), but they actually also hoped to receive the provider’s feedback, otherwise they may think it’s useless for them.”“It would be better if patients’ medication, lifestyle, and complications could be added to the received dashboard.”Some demands which require future consideration:Research in developed countries indicated that mobile technology could be regarded as an effective compliment to chronic disease management to some extent . It is oHypertension is a major health threat in both developed and developing countries ,22, and After training, it was not difficult, and it did not take long for elderly patients with hypertension to use the measurement instrument. From this point of view, it was feasible for patients to complete blood pressure measurements at home.Reminders were used to remind the patient to complete the blood pressure measurement. Even though the specific content of the reminders showed no difference in the patient’s measurement behavior in our study, our results suggest the importance of reminders should not be neglected and this result was similar to a previous study . It mighA promising prospect was found based on the current results of our study: patients had a healthier attitude toward life and gained a better understanding of their blood pressure through the at-home daily measurements during the intervention. Our two-week study of blood pressure monitoring helped most patients develop a habit of monitoring their own blood pressure. Additionally, patients had a positive attitude towards using a similar app in the future, which showed it was feasible to use mobile medical applications in community health care settings in urban China to manage hypertension patients. We also found an important reason for patients compliance with measurements: the hope of improving their health. Hypertension, a chronic non-communicable disease, usually does not show any symptoms  and is tFor patients, it may make it easier for them to adhere to blood pressure measurements if they can make a plan according to their own schedule. For health care providers, this technology provided them with a dynamic and effective way to control patients’ at-home blood pressure measurements ,32, whicAdditionally, and in order to increase the acceptance of a future app for patients in the green category, we believe adding more modules and information may be beneficial such as including patients’ lifestyle, complications, etc. Also, in the patients’ feedback and results, it would be more helpful for providers to offer corresponding diagnostic opinions and suggestions based on the overall situation of the patients. Mobile applications are a useful tool to provide patients with interventions regarding daily behavior and nutrition to reduce blood pressure . A suitaThere were some strengths and limitations of this study. We collected app features and fine-tuned the messages during beta test one and two, in order to ensure patients’ satisfaction with our future app. For example, time intervals between daily reminders were increased to one hour and we found the tone of the messages needed to be modified in beta test one. Based on this and after much discussion, we designed our dashboard to contain personalized reminders and feedback messages for patients . HoweverIn summary, through this mobile health hypertension intervention, we were able to demonstrate the feasibility, uptake and acceptability of the mobile health application for both patients and physicians. As we shift to a digital age, the adoption of mobile-based technology may provide a new approach for monitoring and treating patient’s blood pressure. More testing, modification, implementation and measurement of the effects of such applications on actual blood pressure control are surely needed in order to address the increasing health care burden of hypertension.


Although flavonoid phytoestrogens have been suggested to be associated with reduced risk of colorectal cancer (CRC), their influence on CRC prognosis remains uncertain. A population-based cohort of 2051 patients diagnosed with stage I–III CRC in southwest Germany in 2003–2010 were followed for five years. Post-diagnostic serum concentration of genistein and luteolin were measured using Ultra-Performance Liquid Chromatography with mass spectrometry. Multivariable Cox regression analysis was conducted to calculate the Hazard Ratios (HRs) and 95% confidence interval (CI) for the association between flavonoids concentration and overall morality, CRC-specific mortality, CRC recurrence, and disease-free survival (DFS). Median (interquartile range) serum concentration of genistein and luteolin was 11.90 ng/µL (10.08–14.13) and 7.20 ng/µL (6.40–8.16), respectively. Neither serum genistein nor luteolin was associated with CRC prognosis. There was no clear evidence of departure from linearity. However, the association might be differential by adjuvant chemotherapy. Associations pointed towards lower risk in patients who received chemotherapy and higher risk in those without chemotherapy for overall mortality regarding serum genistein (P-interaction = 0.02) and correspondingly for CRC recurrence (P-interaction: 0.03) and DFS (P-interaction: 0.01) with respect to luteolin. Our study provides little evidence that serum genistein and luteolin are associated with colorectal cancer prognosis. Future studies are warranted to evaluate the potential interaction with adjuvant chemotherapy. Flavonoid phytoestrogens are bioactive, polyphenolic compounds found in plant-based food and share common chemical structural characteristics with endogenous estrogen. There are six subclasses of flavonoid phytoestrogen: Isoflavones, flavones, flavonols, flavanones, flavanols and anthocyanins. Major dietary sources of flavonoid phytoestrogens include fruits, vegetables, tea, wine, grains and herbs ,2.After consumption, flavonoids undergo extensive metabolism. Absorption occurs in both the small and large intestines, with a large portion of flavonoid metabolites reaching the colon . ExposurExperimental studies have shown that flavonoid phytoestrogens have antioxidant, anti-inflammation and anti-cancer properties. Several biological mechanisms are involved in the anti-tumorigenic effect of flavonoids, including anti-angiogenesis, induction of apoptosis, inhibition of tumor cell adhesion and invasion, and estrogenic activities . With a The patient cohort is derived from the DACHS study (Darmkrebs: Chancen der Verhütung durch Screening) which is an ongoing population-based case-control study conducted in southwest Germany from 2003. Details of the design and methods were described previously ,17. BrieComprehensive data of the patients on sociodemographic, lifestyle and reproductive factors and family and medical histories were collected by a trained interviewer through standardized questionnaires at recruitment shortly after diagnosis. Clinical and pathological data were retrieved from the medical records. Three years after diagnosis, treatment details, disease progression and comorbidities information were obtained from their physicians with a standardized questionnaire. At the five-year follow-up, a self-administrated questionnaire was sent to each participant to update their information and collect further data on quality of life, and long-term outcomes including cancer recurrence and comorbidities. Vital status at both three-year follow-up and five-year follow-up was obtained from population registries, and the cause of death was verified by death certificates. This study was approved by the ethics committees of the Medical Faculty of Heidelberg University and the State Medical Boards of Baden-Württemberg and Rhineland-Palatinate. Written informed consent was obtained from all participants.− with electrospray ionization at 0.8 kV capillary voltage and acquisition of individual single ion records using optimized cone voltages (CV): 269 m/z for genistein (CV 20 V) and 285 m/z for luteolin (CV 20 V). The limit of detection for genistein and luteolin was 0.25 pg/µL. Serum concentrations of genistein and luteolin were measured by Ultra-Performance Liquid Chromatography with mass spectrometry (UPLC/MS) at the Centre for Organismal Studies (COS), University of Heidelberg over a period of six months. Blood samples were drawn at the time of the baseline interview shortly after diagnosis, from which multiple aliquots were made and stored at −80 °C until measurement. The extraction approach was adapted from a validated method ,19. BrieInter-batch variation was observed in our study, and the inter-batch coefficient of variation was 61.3% and 44.0% for genistein and luteolin, respectively. It may have been introduced by either the SPE extraction step or the UPLC measurement step. Therefore, a three-step re-calibration method was used to account for the batch effect . First, The paraffin-embedded surgical specimens were requested from the corresponding department of pathology for a subsample of patients. The expression of estrogen receptor beta (ESR2) was determined by immunohistochemistry in the tissue microarray sections using the antibody 14C8 by Abcam  . CpG islFor this analysis, patients at stage I to III with available phytoestrogen measurement and recruited between January 2003 and December 2010 were included. Ten patients without surgery were further excluded from the analysis since surgery is the main treatment for stage I to III CRC and removal of intestine might influence the absorption of phytoestrogen. Multivariate Cox proportional hazard models accounting for late entry were used to evaluate the association of genistein and luteolin with prognosis of colorectal cancer, including overall mortality, CRC- specific mortality, CRC recurrence, and disease-free survival. Re-calibrated genistein and luteolin concentration were modeled both as a continuous variable after log2 transformation and in four categories defined by quartiles. In the latter models, the lowest quartile was used as the reference category. The follow-up period was defined as the interval between the date of study recruitment and the date of censor. Individuals were censored at the date of death, last contact or outcome of interest, whichever came first. Patients who were censored within 30 days after diagnosis were further excluded. Additional covariates were selected based on backward selection at the α-level of 0.2 from the variables including gender, stage, cancer site, body mass index (BMI) category, smoking, alcohol consumption, diabetes, cardiovascular disease (CVD), education, physical activity, detection mode, history of colonoscopy, regular use of nonsteroidal anti-inflammatory drugs, constipation, antibiotics use, red meat intake, adjuvant chemotherapy, radiotherapy, and timing of blood draw. After variable selection, age at diagnosis, gender , stage , cancer site , BMI , education , physical activity , screening detected tumor , adjuvant chemotherapy , CVD , diabetes , constipation , interval between chemotherapy and blood drawn , and interval between surgery and blood drawn  were included in the final model. The analyses were performed with complete cases after exclusion of participants with missing covariates (exclusion <3%). Proportional hazard assumptions were checked for all included variables, and no substantial departure was detected.To investigate potential effect modification, subgroup analyses were conducted by demographic factors , clinical factors , treatment factor (adjuvant chemotherapy), comorbidity-related factors , tumor molecular characterization  and timing of blood draw (with respect to chemotherapy and diagnosis). Subgroup analyses by adjuvant chemotherapy were performed among those with a stage II or III CRC who had the possibility to undergo adjuvant chemotherapy according to the guidelines. Subgroup analyses by tumor molecular characterization were restricted to those for whom tumor tissue was available. The association analysis in female was further adjusted for hormone replacement therapy . The interaction terms were evaluated by likelihood ratio test statistics, comparing models with and without interaction term. To account for the non-linear effect of genistein and luteolin, we further carried out a restricted cubic spline model with three fixed nodes  , which cAll analyses were performed using the SAS statistical software package, version 9.3 . All tests were two-sided with a significant level of 0.05. Of the 2051 patients diagnosed with stage of I to III colorectal cancer, serum genistein concentration was detected in 2029 participants, and luteolin concentration in 2011 participants. The mean age at diagnosis was 68.2 years (standard deviation: 10.6 years). Approximately 40% of the patients were female, and 59% had colon cancer. Blood was drawn shortly after diagnosis : 14–285) and 93% were after surgery . Median (IQR) serum concentration of genistein and luteolin was 11.90 ng/µL (10.08–14.13) and 7.20 ng/µL (6.40–8.16), respectively. The genistein concentration was higher among participants with older age, a history of diabetes or cardiovascular disease and blood drawn before surgery, while luteolin concentration is higher among those with KRAS mutations . OverallAfter a median follow-up of 5.2 years (IQR: 4.4–5.3), a total of 475 patients died (23.2%), of whom 254 (12.5%) died of CRC. Besides, 400 (19.6%) patients had a CRC recurrence. In total, 585 (28.6%) had either deaths or recurrences as events.Multivariable-adjusted HRs for genistein and luteolin concentration in relation to colorectal cancer prognosis are shown in The associations of genistein and luteolin with colorectal cancer prognosis were not modified by age, gender, cancer site, stage, BMI, diabetes, and CVD. However, a statistically significant interaction with adjuvant chemotherapy was observed for both genistein and luteolin among stage II and stage III patients. Serum genistein was associated with better overall survival among those with adjuvant chemotherapy , but with poorer overall survival among those without adjuvant chemotherapy  . For lutNo substantial differences were found according to the timing of blood drawn with respect to diagnosis and chemotherapy for both genistein and luteolin . The higAnalyses by colorectal cancer molecular features including CIMP status, MSI status, KRAS mutation and ESR2 expression showed no effect heterogeneity for either genistein or luteolin . The higIn this large population-based prospective study of colorectal cancer patients, no significant association was found for either serum genistein or luteolin concentration with overall mortality, CRC-specific mortality, CRC recurrence, or disease-free survival. However, there may be differential association according to adjuvant chemotherapy.The results from this study were in line with a previous study of 411 CRC patients conducted in Spain, which is by far the only study that investigated a possible association between colorectal cancer prognosis and phytoestrogen exposure although based on estimated dietary intake. They did not find dietary intake of flavone and isoflavone to be associated with either colorectal cancer recurrence or overall mortality . CompareOur evaluation of potential effect modification for several factors suggest that the association between serum flavonoid phytoestrogens concentration and colorectal cancer prognosis might be differential by adjuvant chemotherapy. Higher serum genistein and luteolin concentrations appeared to be associated with an improved CRC prognosis among patients who had undergone adjuvant chemotherapy and with a poorer prognosis among those who had not, although not all the associations were statistically significant. While this might be a chance finding due to multiple testing, there is some biological plausibility for a beneficial effect of high phytoestrogen levels in patients who received adjuvant chemotherapy. Genistein and luteolin have been found to sensitize chemo-resistant colorectal cancer cells to chemotherapy using 5-fluorouracil ,25, whicOur study did not support a significant association of genistein and luteolin with colorectal cancer prognosis, yet anticarcinogenic effects of genistein and luteolin have been found in experimental studies, particularly mediated through an estrogenic effect. Owing to a similar chemical structure as endogenous estrogen, flavonoid phytoestrogens can bind to ESR2, the predominant estrogen receptor expressed in the colon mucosa, and activate estrogen-responsive pathways ,30, whicIn the present study, flavonoid phytoestrogen levels were quantified by the UPLC/MS method which was adapted from a previously validated method ,19. In cThe strengths of our study include the prospective study design; a large number of colorectal cancer patients; the completeness of follow-up; and a validated method ,19 to meIn conclusion, our study provides limited evidence of an association between post-diagnostic serum genistein and luteolin and the prognosis of colorectal cancer. However, higher genistein and luteolin concentrations may possibly be associated with a better prognosis among those who underwent adjuvant chemotherapy. Future studies with the blood sample drawn and phytoestrogen measured at different time-points and larger sample size are required to confirm the current findings.