The coexistence of raised blood pressure (BP) in people with type 2 diabetes mellitus (T2DM) is a major contributor to the development and progression of both macrovascular and microvascular complications. The aim of our study was to determine the prevalence of uncontrolled BP and its associated factors in persons with T2DM in a district in Kerala.The study was conducted in Ernakulam district in Kerala, and a total of 3,092 individuals with T2DM were enrolled after obtaining consent. Those with a BP “above or equal to 140 mmHg” and/or “above or equal to 90 mmHg” were thus considered to have uncontrolled BP. If the BP was equal or >140 and/or 90 mmHg, a repeat reading was taken after 30 min and the average of the two was considered. Basic demographic details were enquired along with electronic measurement of BP, HbA1c estimation and screening for diabetic retinopathy, peripheral arterial disease (PAD), and peripheral neuropathy. Quantitative and qualitative variables were expressed as mean (SD) and proportions, respectively. The model for determinants of uncontrolled BP was developed adjusting for age, gender, education, duration of diabetes, occupation, body mass index (BMI) and clustering effect.p < 0.001], unemployment , duration of diabetes > 11 years , and BMI ≥23  were found to be independent determinants of high BP levels when adjusted for the aforementioned variables, gender, education, and cluster effect. The association between complications, such as peripheral neuropathy, PAD, and retinopathy showed a higher risk among those with uncontrolled BP. Retinopathy was 1.35 times more , PAD was 1.6 times more , and peripheral neuropathy was 1.5  times more compared to their counterparts.The mean age of the study population was 59.51 ± 9.84 years. The mean duration of T2DM was found to be 11.3 ± 6.64 years. The proportion of uncontrolled HTN adjusted for clustering was 60% (95% CI 58 and 62%). Among them, only one in two persons (53.3%) had a history of hypertension. Age >60 years [adjusted odds ratio (aOR) 1.48, 95% CI 1.24, 1.76; Target BP levels were far from being achieved in a good majority of the persons with T2DM. To reduce further macrovascular and microvascular events among people with T2DM, effective awareness and more stringent screening measures need to be employed in this population. Type 2 diabetes mellitus (DM) is a metabolic disorder characterized by insulin resistance and insulin hyposecretion that result in hyperglycaemia. It is estimated that by the year 2030, about 439 million adults (7.7%) will be affected by diabetes, globally . AccordiHypertension is defined as a condition where the blood vessels have persistently raised pressure. The coexistence of hypertension or blood pressure (BP) above the target level in patients with type 2 diabetes mellitus (T2DM) is a major contributor to the development and progression of macrovascular and microvascular complications . The comBringing down BP has proven to be beneficial in reducing complications associated with diabetes. Several studies have shown that treating hypertension in people with diabetes can reduce atherosclerotic cardiovascular disease (ASCVD) events, heart failure, and microvascular complications . As per However, maintaining the target range of BP is still a challenge. In a European study in 24 countries, a target BP level of <140/90 mmHg was achieved only in 54% of people with diabetes . StudiesA community-based cross-sectional study was carried out in Ernakulam district in Kerala, India. The district, which is an administrative division in the state, has the highest population density and is the commercial capital of the state. A prevalence of 20.6%  was usedA two-stage cluster sampling with population proportionate to size sampling (PPS) was carried out. In the first stage, 33 clusters, which are local self-government areas, were drawn by probability proportional to their size. The population of all the local self-government (LSG) areas was listed. The cumulative population was calculated. The total population was divided by the number of clusters to determine the sampling interval. The first number was picked by the random number table within the sampling interval. The corresponding LSG was selected. The sampling interval was added 33 times to get the 33 LSG areas, which are the clusters. The frontline health worker [accredited social health activist (ASHA)] of each ward provided the list of persons with diabetes to the Primary health centre (PHC). The team at the PPHC chose every third/fourth person from the list. Thus, about 110 persons were provided a referral card and referred considering a non-response rate of 20%.The first 85–90 participants who came to the camp with diabetes for more than a year were enrolled in the study after obtaining informed consent. Local camps were conducted in the selected LSG areas under the aegis of an international non-government organization (NGO), a tertiary care center, Primary Health Center, and National Health Mission. Thus, a total of 3,092 persons with diabetes were enrolled. The inclusion criteria of the study included adults with type 2 diabetes of at least 1 year of duration. The exclusion criteria were those who could not respond to the questions with coherence or those who were cognitively impaired, pregnant woman, and above 80 years. However, in order to efficiently utilize resources, the screening for complications, such as retinopathy, peripheral arterial disease (PAD), and peripheral neuropathy, was carried out among persons with more than 5 years of diabetes. About 33 camps were conducted from November 2020 to March 2021 by a multidisciplinary team of community physicians, ophthalmologists, doctors with training in Podiatry, nurses, laboratory technician, optometrists, and medical social workers. Institutional ethical committee approval was obtained vide IEC-AIMS-2020-COMM-186 dated November 9, 2020.The outcome variable was uncontrolled BP among persons with type 2 diabetes. The BP was considered to be controlled if the systolic and diastolic values were <140 and <90 mmHg. This was also synonymous with having attained target BP. Those with a BP “above or equal to 140 mmHg” and or “above or equal to 90 mmHg” were thus considered to have uncontrolled BP. The BP was measured by the OMRON HEM 7124 automatic blood pressure monitor  by measuring upper arm BP. If a level above or equal to 140 and or 90 mmHg was observed, the measurement was repeated after 30 min and the average of the two readings was taken . SeveralAfter lying down and being made comfortable, the vibration perception threshold (VPT) was tested using a biothesiometer. A probe was placed in the palm of patients to familiarize them with the vibration perception. The patient was advised to feel the vibration on his/her feet and slowly vibration intensity was increased. At the point at which the patient felt the vibration, the VPT was recorded in volts and graded. This indicated the threshold voltage that can be perceived by the person. The probe was applied to the big toe and medial malleoli. The vibration intensity was increased gradually by turning the dial. The VPT value was graded as <15 volts as normal (Grade I), 16–20 volts as mild loss of sensation (Grade II), 21–25 volts as moderate loss of sensation (Grade III), and >25 volts as severe and abnormal (Grade IV) .The patient continued to be in the lying posture, and ankle-brachial pressure index was measured to detect PAD. First brachial BP was measured using a sphygmomanometer and handheld Doppler, then the ankle pressure of each leg was measured, and the ratio of ankle pressure to brachial pressure was calculated for the left and right lower limbs. The BP cuff was placed on the arm, with the limb at the level of the heart. The ultrasound gel was applied in the antecubital fossa over the patient's brachial pulse. The transducer of the handheld Doppler was placed over the antecubital fossa on the gel, and the transducer was positioned to maximize the intensity of the signal. The cuff was then inflated to about 10 mmHg above the expected systolic BP of the patient such that, the Doppler signal disappeared. The cuff was then deflated at approximately 1 mmHg/s. When the Doppler signal re-appears, the pressure of the cuff is recorded as brachial systolic pressure. To measure ankle pressure, the cuff was placed immediately proximal to the malleoli. The ultrasound gel was applied on the skin overlying the dorsalis pedis (DP) artery in the foot. The Doppler signal of the DP artery was found slightly lateral to the midline of the dorsum of the foot. Using a standard handheld Doppler probe and the ultrasound gel, the signal was located. The cuff was inflated till the Doppler signal was no longer heard. Then using the same technique, the cuff was deflated until the Doppler signal re-appeared. The measurement was recorded. The Ankle Brachial Index (ABI) was calculated for each leg. The ABI value was determined by taking the higher pressure of the two arteries at the ankle, divided by the brachial arterial systolic pressure. In calculating the ABI, the higher of the two brachial systolic pressure measurements was used. In normal individuals, there should be a minimal (<10 mmHg) interarm systolic pressure gradient during a routine examination. A reading ≥1.3 was considered to be abnormal vessel hardening, 0.9–1.2 to be normal, 0.50–0.79 to be moderate arterial disease, under 0.50 considered as severe arterial disease .Retinopathy was assessed by mydriatic fundus photography and rechecked by indirect ophthalmoscopy. All patients underwent visual acuity examination with available glass correction and pinhole to see if there was any improvement with a further change of glasses. All patients were dilated with tropicamide eye drops and mydriatic retinal photography was performed. All patients also underwent retinal examination with an indirect ophthalmoscopy by a trained ophthalmologist and retinal findings and diagnosis were confirmed. Grading of diabetic retinopathy was done on site and confirmed with viewing the retinal photographs by experts.p < 0.1 in the univariate analysis was carried out. Age, gender, duration of DM, education, BMI, and occupation were considered as fixed effects, and cluster was considered as random effect in the logistic regression model. Adjusted odds ratio and 95% CI are reported. This was carried out in STATA 15 .The data collected were entered in excel and data analysis was carried out in Statistical Package of Social Sciences (SPSS) , 24. ForThe mean age of the study population was 59.51 years ± 9.84, and it ranged from 29 to 80 years. There was an almost equal distribution among persons less than or equal to 60 years, 1,423 (46.2%) and above 1,652 (53.7%). Men constituted only about a third  of the participants and more than three-quarters [2.422 (78.4%)] of the respondents were from rural areas . HoweverThus, the target BP for persons with diabetes was achieved by only 1,205 (39.9%) patients. Complications, such as PAD and peripheral neuropathy, were found among about a half [738 (48.5%)] and more than a half [963 (53.5%)], respectively. Retinopathy was found among more than a fourth, i.e., 612 (28.9%).p < 0.001). Women had a significantly higher percentage of uncontrolled BP at 61.7% (p < 0.019). Uncontrolled BP was higher among those with a duration of diabetes of more than 11 years (p < 0.001). The uncontrolled BP was found to significantly decrease with the improvement of employment status from 66.2 to 54.6% (p < 0.001). BP was significantly above the target level in those with a BMI ≥23. Others, such as rural-urban residence, education, socioeconomic status, physical activity, Hba1c, heart disease, and respiratory disease, were not found to be significant , duration of diabetes , BMI ≤ 23  were found to be independent predictors of high BP levels when adjusted for the aforementioned variables, education, gender, and cluster  1.48, 95% CI 1.24, 1.76;  cluster . For thep < 0.03), PAD was 1.6 times more , and peripheral neuropathy was 1.5  times more  pandemic and before the second wave started in Kerala. Therefore, it is difficult to ascertain whether COVID may have contributed to higher BP levels.The target levels of BP among people with type 2 diabetes are far from being achieved. This needs emphasis through patient and physician awareness. Increased BP has been associated with micro- and macro-vascular complications, such as retinopathy, neuropathy, and PAD, respectively. Control of BP to below target levels is thus very important for persons with diabetes.The datasets presented in this article are not readily available because permissions will be required from Amrita Institute of Medical Sciences (Kochi), National Health Mission (Ernakulam) and Lions Clubs International (District 318C) for release of data. Requests to access the datasets should be directed to Amrita Institute of Medical Sciences, Kochi.The studies involving human participants were reviewed and approved by IEC Amrita Institute of Medical Sciences, Kochi. The patients/participants provided their written informed consent to participate in this study.AS contributed to study conception, data collection, interpretation of data, and drafting the manuscript. VK contributed to conception, implementation, and editing of manuscript. VMe contributed to conduct, data collection, and editing of manuscript. MM analysed the data. RN contributed to conduct, data collection, and editing of the manuscript. GP contributed to conception, implementation, and data collection. MN contributed to study conception and supported in data collection. JM contributed to conception and editing of the manuscript. VMa contributed to conduct of the study and editing of the manuscript. All authors read and approved the manuscript and critically revised the manuscript for important intellectual content.This work was funded from Lion Club International Foundation (DIA16895/318-C).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


BACKGROUND: Some case-control association studies revealed the relationship between some endothelin-1 (ET-1) gene polymorphisms and blood pressure. Because no report was available about the relationship between any ET-1 gene polymorphism and incidence of hypertension, we examined the relationship between novel ET-1 gene polymorphism (G862T / Ala288Ser in exon 5) and incidence of hypertension by a retrospective cohort study.METHODS: The subjects were Japanese workers at a company in Shimane Prefecture in Japan. The polymorphism with genome DNA extracted from the blood of the workers was analyzed using the polymerase chain reaction confronting two pair primers method. According to the results of two regular health checkups with a 6-year interval, the study population was divided into two groups by blood pressure and antihypertensive treatment in 1998, after excluding people who had hypertension in 1992.RESULTS: There were 133  incidences of hypertension observed among the study population of 922 . In the univariate analysis, odds ratios o Ala/Ser and Ser/Ser against Ala/Ala were 0.98 : 0.7-1.4) and 0.79 (95% CI: 0.4-1.6), respectively. In the multivariate analysis adjusted for sex, age, body mass index, serum total cholesterol, fasting blood sugar, and smoking and drinking habits, odds ratios for Ala/Ser and Ser/Ser against Ala/Ala were 0.97 (95% CI: 0.7-1.4) and 0.75 (95% CI: 0.4-1.5), respectively.CONCLUSIONS: The ET-1 gene polymorphism in this study did not seem to be associated with the incidence of hypertension among the Japanese workers. In fact, increased plasma ET-1 level has been demonstrated among pre-eclamptic pregnant women,6 patients with hypertension7 and diabetes,8 respectively. Furthermore, because a reduction effect on blood pressure with the induction of ET receptor antagonist was observed,9,10 the role of ET-1 in regulation of human blood pressure was confirmed.Endothelin (ET) is known as the most potent vasoconstrictor among reported endothelial-derived vasomotor agents.11,12 Some epidemiologic studies analyzing the relationship between some polymorphisms in this locus and hypertension have been performed as case-control association studies.6,13-18 Moreover, the relationship between a polymorphism in the ET-1 gene and ventricular arrhythmia has been reported.19The human ET-1 gene was assigned to chromosome 6, and spans approximately 5.5 kb with 5 exons and 4 introns.20 the prevalence of the polymorphism among general population was not available. In this way, the presence of a novel human ET-1 gene polymorphism was revealed, but the relationship between the polymorphism and hypertension was not available.Although the presence of a novel polymorphism, the G/T polymorphism with an amino acid substitution (Ala to Ser) at codon 288 in exon 5 at position 862 of the ET-1 gene (Ala288Ser), was released,Thus, in the present study, we analyzed the relationship between the novel ET-1 gene polymorphism and hypertension among Japanese workers. To assess the relationship more detail than in previous reports, a retrospective cohort study enrolling lifestyle factors as considerable variables was performed.The study population was recruited from workers of a company who received annual health checkups and gave written informed consent in 1998, in Shimane Prefecture in Western Japan. The total number of the study population was 1,978. Among the study population of our study, 1,175 subjects were received annual health checkups in both 1992 and 1998. The number of subjects who assessed as normotensive was 975, after excluding hypertensive in 1992, sat as a study cohort. Because there were 922 subjects whose various variables for this study were known, 95% of normotensives in 1992 were enrolled in this study as eligible cohort subject. The incidence of hypertension was assessed at the health checkups in 1998. The following selection criteria for hypertension were applied both in 1992 and 1998: (1) hypertension was defined by systolic blood pressure (SBP) of 140 mmHg and over and/or diastolic blood pressure (DBP) of 90 mmHg and over; or (2) subject received medication for hypertension. The eligible subjects were divided into two groups by incidence of hypertension . The number of subjects who developed the hypertension in 1998 was 133, 14.4% of the eligible subjects. Among the 133 incident cases, 128 subjects were assessed based on their blood pressure in 1998. Further, 111 subjects showed SBP less than 160 mmHg and/or DBP less than 100 mmHg.21 basically. Because it seemed to provide the information of which equal to use the second of two measurements and better than use the mean of two measurements, this method was employed for our studies.Blood pressure was measured with the subjects seated, on the right arm, using a standard mercury sphygmomanometer after at least 5 minutes of rest subsequent to urination. Phase I and phase V Korotkov sounds were recorded as SBP and DBP, respectively. Blood pressure was measured once for each subject, when SBP was less than 140 mmHg and DBP was under 90 mmHg. In case of SBP with 140 mmHg and higher and/or DBP with 90 mmHg and higher, re-measure was performed after at least 5 minute interval. Then the lower of the two measurements was used as the blood pressure. Although this method is not common for epidemiologic studies, it is common for health checkups for workers. The purpose of this method is to evaluate whether each subject has hypertension or not. Further, this method is based on the recommendation from the Japanese Association for Cerebro-cardiovascular Disease control,Body Mass Index (BMI), body weight in kilogram divided by squared height in meter, was used as a measure of body composition.Blood samples were drawn into disposable plastic vacuum tubes  from a peripheral vein. Serum total cholesterol (TC) and fasting blood sugar (FBS) were measured using an autoanalyzer . TC and FBS were determined enzymatically using commercial enzyme kits .Alcohol drinking and smoking habits were assessed using a questionnaire on lifestyle factors.20 according to variation in the exon loci where the protein is encoded. The present study is the first report to examine the association between this polymorphism and hypertension.The ET-1 gene polymorphism studied here was selected from released polymorphisms22 was used to analyze the Ala288Ser polymorphism in the ET-1 gene. The design of 4 primers for PCR-CTPP in the franking region of the G/T polymorphism associated with the Ala288Ser polymorphism in the ET-1 gene was as follows: the forward primer was 5′-CCT CGC TCC CAT TCT AAG CAT AGG G-3′, the reverse primer was 5′-CCT TTG CCA GTC AGG AAC CA-3′, the G allele-specific forward primer was 5′-GAT CCC AAG CTG AAA GGC AAG-3′, and the T allele-specific reverse primer was 5′-TCA CAT AAC GCT CTC TGG AGG GA-3′. PCR was performed in a thermal cycler . The total volume of PCR reaction solution was 10 ml for each sample, containing 2 mmol/L of each primer. The solution contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 250 mmol/L of each dNTP, and 5U of AmpliTaq Gold DNA polymerase . For the PCR procedure, initial denaturation for 10 min at 95°C was subsequent to a hot start at 95°C. Following the PCR cycle with denaturation for 30 sec at 95°C, annealing for 30 sec at 65°C, and polymerization for 60 sec at 72°C was performed 35 times. Samples for electrophoresis were prepared with 4.0 ml of PCR product and 0.4 ml of Loading Buffer . The samples were electrophoresed in 6% polyacrylamide gels and DNA was visualized by ethidium bromide staining for genotype assessment.Genome DNA was extracted using a fully automatic nucleic acid extractor . The extracted genome DNA was stored at -20°C until genotyping. The polymerase chain reaction confronting two pair primers method (PCR-CTPP)® software  was employed for all statistical analysis. Unpaired analysis of results by a Wilcoxon rank sum test was performed to assess whether differences of quantitative variables between normotensives and hypertensives were significant. Chi-square tests were calculated to evaluate the distribution and trend of sex, lifestyle and genotype. The odds ratios (OR) and 95% confidence intervals (CI) of each factor for incidence of hypertension were estimated by univariate and multivariate unconditional logistic regression analysis. Statistical significance was defined as p<0.05.The SAS23-27 Informed consent of gene analysis was obtained from each study subject.The present study was approved by the Ethics Committee of the Faculty of Medicine, Tottori University .Sex distribution, mean value and standard deviation (SD) of age and various quantitative variables, and distribution of lifestyle factors at the health checkups in 1992 by incidence of hypertension in 1998 are shown in The distributions of genotype and allele frequency are shown in The relationships between genotype, allele frequency and incidence of hypertension estimated by unconditional logistic regression analysis are shown in The distribution of the Ala288Ser polymorphism in the ET-1 gene among Japanese workers was revealed by the present study; the Ala/Ala, Ala/Ser, and Ser/Ser genotypes were in 49%, 41%, and 10% of subjects, respectively. The Ser allele frequency showed a high proportion at 30%. On the other hand, with both univariate and multivariate analysis adjusted for considerable variables, the polymorphism showed no statistical significance for the incidence of hypertension.13 Stevens et al.14 and Brown et al.16 studied on single base insertion of adenine at position 138 in the ET-1 gene, and showed the association between the polymorphism and DBP among control subjects with normal blood pressure. The remaining four reports studied the Lys198Asn polymorphism in the ET-1 gene. Among them, a study of pregnant women revealed that the Lys/Asn and Asn/Asn genotypes significantly raised SBP compared to the Lys/Lys genotype during pregnancy, and the effect disappeared after delivery.6 Other three studies on the polymorphism were population-based.15,17,18 These three reports demonstrated that a steeper increase in blood pressure with BMI occurred in obese subjects with this polymorphism, without distinction of race.As far as we know, seven studies that assessed the relationship between ET-1 polymorphism and hypertension have been published. The first study reported no effect for a Taq1 restriction fragment length polymorphism on hypertension and blood pressure level.There have been no reports available on the Ala288Ser polymorphism in the ET-1 gene, which we studied here, yet. A high proportion of 30% of the Ser allele frequency in this polymorphism was observed. Although no association between the polymorphism and incidence of hypertension was observed after adjusting for confounding factors including lifestyle variables, additional studies are needed to examine the mean of this polymorphism.28,29 It is also known that a higher concentration of serum insulin is observed in obese subjects.30,31 Thus, FBS was included in the analysis to adjust for the confounding factor of insulin resistance of subjects. Moreover, in the epidemiologic knowledge, chronic exposure to smoking would not increase the risk of incidence of hypertension.32 Regarding plasma ET-1 levels, some reports have demonstrated an increase by smoking,33,34 whereas another has reported a decrease.35 On the other hand, alcohol drinking has been shown to increase the risk of incidence of hypertension.36 Regarding plasma ET-1 levels, one study has demonstrated no induction by alcohol drinking.37 The polymorphism studied in this report did not seem to be a candidate gene responsible for incidence of hypertension because it showed no significant relationship after adjusting for these lifestyle variables.The relationship was analyzed, and adjusted for FBS and lifestyle variables as well as sex, age, BMI, and TC, in this study. Participation of insulin in the modulation of plasma ET-1 levels was suspected.38 Second, the influence of our definition of incidence of hypertension. Although 133 subjects developed hypertension in 1998, many of them were assessed based on their blood pressure and showed mild hypertension. The Blood pressure was measured carefully, however, because of the individual variation and other biases39 in blood pressure, there is possibility that our definition of incidence of hypertension leads the overestimation of the number of incident cases and results the underestimation of the relationship between the polymorphism and incidence of hypertension. Third, the relationships among study subjects were not confirmed. The possibility that relations and siblings showed similar characteristics and influenced the results cannot be denied. Fourth, the accuracy of the record of health checkups is listed. Lifestyle variables were surveyed, through interview by public health nurses, based on a self-statement by each subject. The presence of recall bias is suspected. A prospective cohort study should be performed for a more exact evaluation.All the reports which have studied the relationship between polymorphism in the ET-1 gene and hypertension were analyzed using case-control association studies. A retrospective cohort study among workers, not hospital-based, was employed in this study. Although a cohort study was employed to reduce the effect of biases, compared to the case-control study, some limitations remain in this investigation. First, for the incident cases of hypertension in this study, we are not able to deny the possibility that they included cases of secondary hypertension. However, the influence of this limitation is thought to be small, since more than 90% of Japanese hypertension cases are essential hypertension.In our results, in neither univariate analysis nor multivariate analysis after adjusting for considerable variables did the Ala288Ser polymorphism in the ET-1 gene show a statistically significant relationship with incidence of hypertension. The Ala288Ser polymorphism in the ET-1 gene did not seem to be a candidate gene responsible for incidence of hypertension among these study subjects.


ChAT), whereby genetic ablation of ChAT in these cells produces hypertension, indicating that production of ACh by TChAT regulates blood pressure. Accordingly, we reasoned that increasing systemic ChAT concentrations might induce vasodilation and reduce blood pressure. To evaluate this possibility, recombinant ChAT was administered intraperitoneally to mice having angiotensin II-induced hypertension. This intervention significantly and dose-dependently decreased mean arterial pressure. ChAT-mediated attenuation of blood pressure was reversed by administration of the nitric oxide synthesis blocker l-nitro arginine methyl ester, indicating ChAT administration decreases blood pressure by stimulating nitic oxide dependent vasodilation, consistent with an effect of ACh on the endothelium. To prolong the half life of circulating ChAT, the molecule was modified by covalently attaching repeating units of polyethylene glycol (PEG), resulting in enzymatically active PEG-ChAT. Administration of PEG-ChAT to hypertensive mice decreased mean arterial pressure with a longer response duration when compared to ChAT. Together these findings suggest further studies are warranted on the role of ChAT in hypertension.Acetylcholine (ACh) decreases blood pressure by stimulating endothelium nitric oxide-dependent vasodilation in resistance arterioles. Normal plasma contains choline acetyltransferase (ChAT) and its biosynthetic product ACh at appreciable concentrations to potentially act upon the endothelium to affect blood pressure. Recently we discovered a T-cell subset expressing ChAT (TThe online version contains supplementary material available at 10.1186/s10020-021-00380-6. Approximately 100 million adults in the United States and more than 1 billion individuals worldwide have hypertension  which induces the release of nitric oxide (NO), termed endothelium derived relaxing factor or EDRF . Mice were singly housed after implanting blood pressure telemeters. ChAT and PEG-ChAT were administered intraperitoneally.Mice were induced under 2.5% isoflurane and implanted with HD-X10 blood pressure telemeters  according to the manufacturer’s instructions. Briefly, mice were anesthetized and placed in dorsal recumbency. A small incision was made in the neck and the left carotid artery was isolated from the surrounding tissue. A hollow catheter was placed in the left carotid artery and advanced to the aortic arch. Once in place, the catheter was secured to the carotid artery with a suture knot. The telemeter body was placed in a subcutaneous pocket on the animal’s flank, and the animal was recovered for 7 days before blood pressure measurements were taken. Surgeries were performed by JAX Surgical Services .Osmotic pumps  were loaded with angiotensin II (Fisher Scientific) solution to produce the desired flow rate (700 ng/kg/min) according to the manufacturer’s instructions and incubated in sterile saline at 37 °C for 48 h before implantation. To implant the pump, mice were induced under 2.5% isoflurane anesthesia and a 1 cm midscapular incision was made. The pump was inserted into the midscapular incision and advanced away from the incision site. The incision was closed with a wound clip or sutures. After 24 h of recovery, blood pressure measurements resumed. After 28 days post-implant, the implants were removed according to the manufacturer’s instructions.N-histidine tag was expressed in E. coli BL21-Gold (DE3) cells. Bacteria were cultured in 2xYT media to an A600 of 0.9. IPTG was added to a final concentration of 3 mM to induce recombinant ChAT production. Five hours later, cells were harvested, re-suspended in cold binding buffer  and subjected to sonication at 4 °C. Supernatant was collected by centrifugation at 13,000×g for 20 min and applied onto a 5-ml high affinity Ni-charged column pre-equilibrated with binding buffer. Following sequential washings with the binding buffer, washing buffer 1 , and buffer 2 , 10% glycerol, pH 7.5), the recombinant histidine-tagged ChAT protein was eluted with 0.5 M imidazole, 10% glycerol, in 0.2× PBS. The recombinant ChAT was further purified by dialysis at 4 °C in buffer containing 0.2× PBS, 10% glycerol and 0.5 mM TCEP (tris(2-carboxyethyl)phosphine), and extensive Triton X-114 extraction to remove contaminating endotoxins. PEGylation was performed using an unbranched amine-reactive MS (PEG)12 reagent (Thermo Scientific), in a buffer containing 10% glycerol, 0.2× PBS, 0.5 mM TCEP at a molar ratio of ChAT protein to MS (PEG)12 of 1–200. Following PEGylation, the protein was then dialyzed in buffer and further extracted with Triton X-114 to remove contaminating endotoxins.Recombinant human ChAT corresponding to residue 119–748  with a Activity of recombinant and PEGylated recombinant ChAT was analyzed using an adapted version of the colorimetric assay developed by Kumar et al. . Brieflyggplot2 and patchwork. Groups were compared using one-way ANOVA with Dunnett’s correction, two-way ANOVA with repeated measures with Sidak’s correction, or paired t-test where indicated. P values < 0.05 were considered significant.Data were analyzed using Graphpad Prism 7.0, Microsoft Excel, DSI Ponemah 6.4, and R. Blood pressure was sampled at 500 Hz and aggregated into 10 s bins to calculate SBP, DBP, MAP, and heart rate. Temperature and activity data were separately averaged over 10 s bins. When averaged over 12 h, data are first binned in 10 s bins, and 12 h of bins are averaged together Figs. D, 4B. WhA widely used murine preclinical model of essential hypertension is administration of angiotensin II (Ang II) . MAP increases significantly within 24 h of Ang II pump implantation during both light cycle and dark cycle Fig. C, MAP duEscherichia coli expression system. As expected from the cDNA sequence for the R isoform (UniProt P28329-3), purified ChAT has an observed migration on electrophoresis of approximately 72 kDa  , and measured blood pressure following administration of ChAT. Pre-treatment with L-NAME significantly attenuates ChAT-mediated decrease in blood pressure . This method is a useful strategy to enhance the pharmacokinetic properties of therapeutic proteins as it increases protein’s hydrodynamic radius and decreases immunogenicity and proteolysis , which corresponded to the time of the day with highest blood pressures Fig. B. In theHere we report that administration of PEG-ChAT to mice with Ang II-induced hypertension significantly attenuates increases in MAP, SBP and DBP without altering other physiological parameters including HR, temperature and activity. These findings reveal the previously unrecognized potential use of exogenous ChAT as an experimental therapeutic in hypertension. Chronic infusion of Ang II in experimental animals induces a gradual increase in blood pressure  are required to maintain normotension in mice via a NO-dependent mechanism. Ablation of ChAT in CD4 T cells leads to chronic hypertension indicating that ChAT expression by T cells is required to deliver ACh to endothelium devoid of direct cholinergic innervation  or vehicle administration, and area under the curve is calculated from a baseline of 100%. Negative values correspond to decrease in SBP. Data are represented as individual mouse data points, mean ± SEM. n = 7–11 per group. ***p < 0.001, one-way ANOVA with Dunnett’s correction. B change in normalized DBP in the 3 h period after PEG-ChAT administration. SBP is normalized to the 30-min period before PEG-ChAT (0.1 or 1 mg/kg) or vehicle administration, and area under the curve is calculated from a baseline of 100%. Negative values correspond to decrease in DBP. Data are represented as individual mouse data points, mean ± SEM. n = 7–11 per group. **p < 0.01, one-way ANOVA with Dunnett’s correction. Figure S7. PEG-ChAT administration does not induce long-term changes in heart rate, body temperature, or activity levels. A HR, B temperature and C activity in the 24 h period post PEG-ChAT administration. Gray background indicates dark cycle. Data are averaged every hour and represented with lines as group mean ± SEM. ns not significant, two-way ANOVA with Sidak’s multiple comparisons test. Figure S8. PEG-ChAT administration during dark cycle induces decreases in SBP and DBP in the following light cycle. A mean SBP, B mean DBP in light cycle. PEG-ChAT was administered at the middle of the dark cycle (6 h), and MAP is averaged 6–18 h post-PEG-ChAT administration. Data are represented as individual mouse data points, mean ± SEM. Vehicle: n = 12, PEG-ChAT 0.1 mg/kg: n = 20, PEG-ChAT 1 mg/kg: n = 21, *p < 0.05, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Dunnett’s correction.


Asthma is a chronic disease characterized by airway hyperresponsiveness, inflammation and mucus production. In Type 2 asthma, two phenotypic components are often co-expressed . Elevated biomarker levels, such as eosinophils (EOS), fraction of exhaled nitric oxide (FeNO) and immunoglobulin E (IgE), are key clinical indicators of Type 2 inflammation. Dupilumab has been recently approved for the treatment of uncontrolled severe Type 2 asthma. Type 2 asthma includes allergic and/or eosinophilic phenotypes. The aim of this analysis was to estimate the dupilumab-eligible population in Italy and characterize it by expected biomarker status.A 4-step approach was carried out to calculate dupilumab-eligible population. The approach consisted in: (1) estimating the total number of asthma patients in Italy ; (2) estimating the number of severe asthma patients with poorly controlled or uncontrolled disease ; (3) stratifying the severe uncontrolled population by biomarker levels  according to the outcomes of the QUEST trial ; (4) identifying the sub-populations of severe uncontrolled asthma patients characterised by raised blood EOS and/or FeNO level (thus indicated to receive dupilumab).According to these estimates, about 3.3 million asthmatic patients live in Italy (6.10% of the population). Of them, almost 20 thousand  have uncontrolled severe asthma. Dupilumab-eligible patients would be N = 15,988, corresponding to 80.1% of the total uncontrolled severe population. Most of these patients  have at least an increase of EOS level, while slightly more than half  have raised levels of both biomarkers. Increased FeNO levels without increased EOS are observed less frequently .There is a strong rationale for testing all asthma biomarkers during diagnosis and disease follow-up. Given the large availability and the limited costs, these tests are cost-effective tools to detect severe Type 2 asthma, stratify patients by phenotype, and drive appropriate treatment decisions.The online version contains supplementary material available at 10.1186/s12948-021-00146-9. Asthma is a chronic disease affecting people of any age characterized by airway hyperresponsiveness (AHR), airway obstruction and inflammation, intermittent airflow and mucus production , 2. WheeThe way asthma has been defined and classified has changed over time. In the past, asthma was considered “a single entity” disease, characterized by abnormal response of T-helper cell Type 2 (Th2) cells and B cells . Today, The pathophysiology of asthma is characterized by the immune response of two CD4 + T-cell subsets, Th1 and Th2 cells. Specifically, the Type 2 asthma endotype (in the past known as “T2-high”) is originated from the complex mechanisms of Type 2 inflammation where two phenotypic components are co-expressed . Type 2 inflammation is mainly driven by Th2 cell activation, which triggers an abnormal generation of cytokines , in response to the detection of different agents  , 8. IndeIL-4 and IL-13 are relatively upstream players in the inflammatory cascade, and they are responsible for various pro-inflammatory activities. IL-13 stimulates the production of eotaxin 1 in airway inflammatory cells, causes airway smooth muscle and goblet cell hyperplasia, transforms fibroblasts into myofibroblasts, increases tracheal-bronchial mucus secretion, collagen production by fibroblasts and subepithelial basal membrane thickening, which are all features of airway remodelling . IL-4 plType 2 is probably the most common type of asthma: about 50%–70% of people with asthma have an underlying Type 2 inflammation , 10, 11.Elevated biomarker levels, such as EOS, FeNO and IgE, are key clinical indicators of Type 2 inflammation , 19. EOSToday, clinical guidelines recommend testing severe asthma patients for multiple biomarkers, i.e. blood and sputum EOS, FeNO, IgE  especially in presence of refractory disease with underlying Type 2 inflammation . In thisEvaluation of Type 2 inflammation biomarkers is of critical importance to guide treatment decision in severe asthma which is refractory to conventional medical therapy. The standard of care of asthma is based on inhaled corticosteroids (ICS), either as monotherapy or in combination with other treatments, such as long-acting β2-adrenoreceptor agonists (LABAs) and/or cysteinyl-leukotriene type 1 receptor antagonists (LTRAs) . However“treatment of adults and adolescents 12 years and older as add-on maintenance treatment for severe asthma with Type 2 inflammation, characterised by raised blood EOS and/or raised FeNO” [Over the last 15–20 years, several biologic therapies have been developed to address the unmet medical need in severe asthma, and many others are under evaluation in clinical trials . Treatmeed FeNO” . Therefoed FeNO” –36. The Stratification of patient population by biomarkers to identify the right eligible patients is a crucial task in the “biologic-era”. While personalized treatment of asthma is producing significant benefits for patients, asthma management costs are increasing. Notably, about 50% of the global asthma budget is allocated to severe patients  , 38.The need for managing resources appropriately and controlling therapeutic expenditure makes biomarker testing even more important, for budget allocation purposes and cost-effective use of high-cost drugs, such as biologics. Quite recently, many studies have been conducted with the objective of estimating the epidemiological and clinical burden of severe asthma in Italy –41. In tThis paper aims to calculate the number of Type 2 severe asthma patients who would be eligible for dupilumab treatment in Italy.A 4-step approach was used: (1) estimation of the total number of asthma patients ; 2) estimation of the number of severe asthma patients, who are poorly controlled or uncontrolled ; (3) stratification of the severe uncontrolled asthma population, by biomarker levels; (4) identification and estimation of the sub-populations of severe uncontrolled asthma patients eligible for treatment with dupilumab, based on appropriate biomarker levels. The methodological approach flow is shown in Fig.  estimatiTo estimate the number of patients affected by asthma in Italy, a prevalence rate of 6.1% was used. This estimate was gathered from the 2016–2017 Italian-adapted GINA  guidelines , and refThe second step of the analysis was to extract the population with severe uncontrolled asthma from the overall patient population with asthma. For this purpose, two different sources were used and then compared to each other: (1) a recent publication from Pedrini et al., 2017 ; (2) theIn their study, Pedrini et al. retrospectively analysed administrative data concerning adult patients (≥ 18 years old) registered in an Italian healthcare system database (the Accounting and Reporting Console -ARCo- database) . Over thThe regional analysis conducted in Veneto adopted stricter requirements  to identify more appropriately patients with severe refractory asthma requiring regular specialist monitoring . The anaThe third step of the analysis was to stratify the severe uncontrolled asthma population by biomarker levels. Stratification of Type 2 asthma by biomarker status is important to identify patients with blood EOS < 150 cells/μL or FeNO < 25 ppb, who must be excluded from patient estimation. For this purpose, the QUEST trial  was considered an appropriate source to evaluate the distribution of patients by Type 2 inflammation biomarkers: EOS, FeNO and IgE . Figure In general, the three largest groups were: (i) patients with increase of both EOS and FeNO levels (EOS ≥ 150 cells/μL and FeNO ≥ 25 ppb) and allergic ; (ii) patients with increase of both EOS and FeNO levels (EOS ≥ 150 cells/μL and FeNO ≥ 25 ppb) and non-allergic ; (iii) patients with EOS ≥ 150 cells/μL, FeNO < 25 ppb and allergic .According to baseline patients’ characteristics of the QUEST trial, two patient groups were considered non-eligible to dupilumab: (i) patients with EOS < 150 cells/μL, FeNO < 25 ppb and allergic ; (ii) patients with EOS < 150 cells/μL, FeNO < 25 ppb and non-allergic . In conclusion, the QUEST trial indicates that 80.1% of patients with severe uncontrolled Type 2 asthma (100.0%-10.5%-9.4%) would have raised blood EOS and/or raised FeNO and would be then eligible for dupilumab treatment.The input data collected through the 4-step approach were used to calculate the number of dupilumab-eligible patients. According to these estimates, about 3.3 million asthmatic subjects live in Italy Table ; line 2.On average, the number of patients with QUEST-like characteristics would be N = 19,960. This number accounts for ~ 6% of all asthmatic patients who receive some regular follow up (i.e. patients with spirometry) and ~ 13% of all actively treated asthmatic patients (i.e. patients receiving ICS).Table Finally, Fig. The present analysis is an attempt to estimate the epidemiological burden of asthma, and to characterize patient population by severity, type of inflammation and expected distribution of biomarker levels in severe patients. We used data from the QUEST trial to estimate the number of patients with raised EOS and/or raised FeNO to identify the subgroup of patients that would be eligible for dupilumab treatment. The recent administrative database analyses , 41 wereFinally, these analyses showed that patients with severe uncontrolled asthma would be ~ 6% of all asthmatic patients who receive some regular follow up (i.e. patients with spirometry) and ~ 13% of all actively treated asthmatic patients (i.e. patients receiving ICS). These figures are quite consistent with published literature, concluding that 5%–10% of patients have a severe form of asthma which is refractory to conventional steroid medical therapy .The QUEST study  was usedPatient distributions, by individual biomarker levels at baseline, were consistent in the QUEST  and the According to our estimates, about 20 thousand patients with severe uncontrolled asthma live in Italy, and about 16 thousand patients (~ 80%) have a Type 2 endotype with raised EOS and/or FeNO, who would make them potentially eligible to dupilumab. Whilst most of dupilumab-eligible patients demonstrate raised EOS level , there is still a non-negligible number of patients  that have increased FeNO levels without increased EOS. This suggests the importance of testing both the biomarkers, during the diagnosis of Type 2 asthma and in the disease follow-up. Indeed, some patients may have clinically uncontrolled asthma but the biomarker expression could be suppressed by pharmacological treatment .Plausibly, there are a few methodological limitations in this analysis, that might affect validity of findings and increase uncertainty of the estimates. First, we used administrative database analyses, rather than clinical registry data, to estimate the overall population with uncontrolled asthma. This might lead to underestimation of patients, as administrative databases do not identify patients: (i) seeking private care; (ii) with intermittent disease, who would temporarily be “out of the system” but would likely reappear later in time. It is estimated that 5%–10% of all asthma patients develop severe refractory disease , 47. TheAccording to GINA guidelines, clinical assessment of severe asthma is a crucial step for disease characterization, as it ensures identification of patients requiring urgent healthcare interventions and highly effective treatments. Biomarker assessment is essential to identify the expression of Type 2 inflammation. According to GINA guidelines and prescribing conditions defined by regulatory agencies, the increase of at least one of the severe Type 2 asthma biomarkers would justify the use of biological therapy. However, in clinical practice, it is important to understand the complexity of the inflammatory mechanisms of Type 2 inflammation for each patient, to choose the treatment with the highest probability of acting against the different Type 2 inflammatory components expressed or co-expressed by the patient. Testing for more biomarkers simultaneously, is strongly recommended during baseline patient assessment and disease follow-up, to identify the possible targets of biological therapy . As a matter of fact, patients with normal EOS but increased FeNO have similar disease severity  as patients with raised EOS and normal FeNO, or patients with both raised EOS and FeNO. In a post-hoc analysis of the QUEST study , the effIn conclusion, it was possible to estimate the number of dupilumab-eligible patients in Italy, using data on clinical assessment and biomarker testing from local studies and QUEST trial . There is a strong rationale for simultaneously biomarker testing (including FeNO) during diagnosis and diseases follow-up. With relatively low-cost tests, physicians can estimate the number of patients with severe asthma with Type 2 inflammation (Type 2 asthma), stratify them by phenotypes , identify the optimal treatment strategy and prescribe biologic therapy appropriately.Additional file1 :Table S3. Codes considered for patient inclusion in the Veneto study


This study aimed to develop a machine learning (ML)-based model for identifying patients who had a significant coronary artery disease among out-of-hospital cardiac arrest (OHCA) survivors without ST-segment elevation (STE). This multicenter observational study used data from the Korean Hypothermia Network prospective registry (KORHN-PRO) gathered between October 2015 and December 2018. We used information available before targeted temperature management (TTM) as predictor variables, and the primary outcome was a significant coronary artery lesion in coronary angiography (CAG). Among 1373 OHCA patients treated with TTM, 331 patients without STE who underwent CAG were enrolled. Among them, 127 patients (38.4%) had a significant coronary artery lesion. Four ML algorithms, namely regularized logistic regression (RLR), random forest classifier (RF), CatBoost classifier (CBC), and voting classifier (VC), were used with data collected before CAG. The VC model showed the highest accuracy for predicting significant lesions (area under the curve of 0.751). Eight variables  were significant to ML models. These results showed that ML models may be useful in developing early predictive tools for identifying high-risk patients with a significant stenosis in CAG. Coronary artery disease is the main cause of out-of-hospital cardiac arrest (OHCA) . ImmediaNewer computational methods, namely machine learning (ML), may allow more accurate prediction than risk assessment tools developed using standard methods. Targeted ML algorithms triggered by patient data have been increasingly developed as clinical decision support tools in various diseases, including sepsis, gastrointestinal bleeding, and cardiac arrest ,6,7. GivThis multicenter prospective observational study used data from the Korean Hypothermia Network prospective registry (KORHN-PRO) 1.0 gathered between October 2015 and December 2018. KORHN is a multicenter clinical research consortium for targeted temperature management (TTM) in South Korea. Twenty-two academic hospitals participated in KORHN-PRO. The study included an informed consent form approved by all participating hospitals, and the study was registered at the International Clinical Trials Registry Platform (NCT02827422). Written informed consent was obtained from all patients′ legal surrogates.The inclusion criteria of KORHN-PRO were as follows: OHCA regardless of etiology of cardiac arrest, age older than 18 years, unconsciousness  after ROSC, and treatment with TTM. The exclusion criteria were as follows: active intra-cranial bleeding, acute stroke, known limitations in therapy and a do-not-attempt resuscitation order, known prearrest cerebral performance category (CPC) 3 or 4, known disease making 6-month survival unlikely, and body temperature < 30 °C on admission. Data were entered into a web-based electronic database registry using a standardized registry form. Each participating hospital had a designated research coordinator responsible for ensuring data accuracy.For the present trial, we excluded patients with obvious non-cardiac causes and STE on the initial ECG from KORHN-PRO. This study used 37 independent and one dependent variable described below from the registry: age, sex, comorbidities  or stroke, hypertension, diabetes mellitus, pulmonary disease, neurologic disease other than cerebrovascular accident (CVA), chronic kidney disease, malignancy, previous PCI, previous coronary artery bypass grafting, and previous ischemic heart disease), family history , resuscitation variables , post-ROSC variables (immediate ECG findings and laboratory findings), and extracorporeal life support. OHCA survivors were defined as OHCA patients who had a return of spontaneous circulation and were admitted to the hospital for management of post-cardiac arrest care including TTM. The outcome variable of this work was whether OHCA survivors without STE had a significant coronary artery lesions. Significant coronary artery disease was defined by invasive CAG as >50% stenosis of the left main stem or >70% stenosis in a major coronary vessel.Through automated ML procedures, four ML algorithms (regularized logistic regression (RLR) , random Logistic regression (LR) is a statistical model that uses the logistic function as a link function to explain the relationship between independent and dependent variables. In the field of ML, an arbitrary constant is added to the cost function of LR and used as a model for classifying unseen data. When learning a model with training data, the constant helps to optimize between the bias and variance by preventing overfitting. LR with this constraint is called RLR, and the coefficients of explanatory variables in the optimized model represent the feature importance of each variable.RF is known as an algorithm that can create an optimal model for prediction or classification in a short learning time from a tabular data structure in which continuous and categorical variables are mixed. In particular, it is possible to prevent overfitting by extracting part of the data through sampling and generating a number of mutually independent decision trees using only some variables that greatly reduce the cost function, predicting the outcome through majority voting and evaluating the importance of the independent features. These are the reasons we adopted RF in this study.In our data, as most independent variables are categorical variables and most of the categorical independent variables are binary, it is essential for algorithms to handle binary categorical variables well. RF may have difficultly generating optimized models if many binary categorical variables are included because it creates a model in a deep tree structure with variables having only one splitting point. As the name suggests, CatBoost can effectively handle categorical variables through sequentially encoding categorical variables or combining highly correlated categorical variables. In addition, because CatBoost uses the boosting algorithm, it is possible to generate an optimized decision tree model in a way different from that of RF.The three algorithms with different strengths and weaknesses create each optimized model. Finally, the voting algorithm was used to combine the prediction probabilities of the models with several voting methods to form an ensemble model.Multiclass classification by splitting a minor outcome group into two subgroups was conducted to enhance the performance of the models. Additionally, analyses were carried out by feature selection by permutation  or the dp-values ≤ 0.05 indicated statistical significance.Continuous variables are presented as the means with the standard deviation or as median values with interquartile ranges (IQRs). Categorical variables are presented as frequencies and percentages. For patient characteristics and comparisons between groups (significant lesion and without significant lesion), we used Student′s t-test for normally distributed continuous variables and the Mann–Whitney U test for nonparametric cases. Fisher′s exact test was used for cases of low-frequency categorical variables, and the chi-square test was used otherwise. In our dataset, nine variables had missing values, and Little’s missing completely at random test  was run During the study period, a total of 1373 OHCA patients (>18 years) treated with TTM were enrolled in KORHN-PRO . Among tAmong them, 127 patients (38.4%) had a significant coronary artery lesion. The four ML models were trained to identify patients who had a significant coronary artery disease among OHCA survivors without STE. The AUROC (95% CI) values of the trained RLR, RF, CBC, and VC models were 0.734 (0.722–0.746), 0.737 (0.729–0.745), 0.736 (0.726–0.746), and 0.751 (0.740–0.762), respectively . The AURThe mean and 95% CI of loss and metrics values were estimated with the 150 models made by a combination of 30 imputed datasets and a five-fold cross validation procedure, and the results are shown in The learning curves for Cohen’s kappa, AUROC, and F1-score of the VC, CBC, RF, and RLR models as a function of the number of sample data are shown in x-axis in The coefficients of feature importance were respectively estimated by the (a) mean of coefficients of RLR models, (b) Gini impurity-based method of the RF model, and (c) prediction values changed algorithm of CBC models . Note thThe global feature importance for the VC model, the best-performing model in this study, was calculated using Shapley values to reliably select important variables to identify patients with a significant coronary artery lesion. In In this study, we developed models based on ML algorithms to predict patients with a significant coronary artery lesion requiring early PCI through only basic patient information before admission. We found that the VC model showed the highest accuracy for predicting a significant lesion (AUROC of 0.751). We also found that eight easily captured variables  were significant.Coronary artery disease is particularly prevalent in cardiac arrest patients with shockable rhythm: up to 96% of patients with STE on their post-resuscitation ECG and up to 42% for patients without STE ,31. Our Considering that the benefit of CAG is associated primarily with providing PCI, identifying patients who have a significant coronary occlusion is crucial. Waldo et al. developed a risk prediction model for the presence of an acute coronary lesion among patients resuscitated from an arrest . They foFor now, except those with STE or cardiogenic shock, the specific subgroups that would benefit from early CAG remain unknown ,3. A stuThe data that accumulate in a stressful emergency room are affected by several biases, including typos and discontinuous laboratory measurement. They have great impacts on ML studies using data made up of already sculpted tabular-type variables. However, it was possible to create better performing VC models that synthesized the effective RLR, RF, and CBC models that were complementary with advantages of different algorithms. The soft voting method, the use of the arithmetic mean of prediction probabilities from the RLR, RF, and CBC models, worked well in this study.The aim of this research was to determine which variables greatly hint at the presence of significant coronary artery lesions. Feature importance determined using Shapley values considering all possible combinations of variables in the VC model can be considered the most reliable result. This provides guidelines to practitioners in emergency rooms on the need to carefully monitor patients with the following: older age, male, initial shockable rhythm, shorter total collapse duration, higher glucose and creatinine, and lower pH and lactate. This result can also be used to prioritize which variables in data to collect by investing limited emergency room resources.As shown in This study has several limitations. There is an important concern about the population selected. KORHN-PRO did not have a solid indication of CAG except STE or cardiogenic shock. The selection of the patients who had undergone CAG was done by the treating physicians, which means there was already a high suspicion that these patients had ACS, creating selection bias. Other issues include the requirement of a computer implementation for the ML model and the limited power to detect other clinical characteristics that may be associated with a significant coronary artery disease due to the modest sample size. Moreover, to appropriately select patients that would benefit from CAG, thrombotic occlusion or culprit lesion was more useful than a significant coronary artery disease. Lastly, there may be valuable variables such as results of echocardiography not included in our registry. Therefore, our VC model is not complete enough to be used in the clinical field right now. Further larger studies with other variables will be needed.ML models may be helpful early predictive tools for identifying high-risk patients with a significant stenosis in CAG. However, further investigations with large datasets including other variables are warranted to improve the prediction model, which can support clinicians’ decisions on early CAG in OHCA survivors without STE.


The recent consensus by the Perioperative Quality Initiative (POQI) on intraoperative hypotension (IOH) stated that mean arterial pressure (MAP) below 60–70 mmHg is associated with myocardial infarction (MI), acute kidney injury (AKI), death and also that IOH is a function of not only severity but also of duration. However, most of the data come from large, heterogeneous cohorts of patients who underwent different surgical procedures and types of anaesthesia. We sought to assess how various definitions of IOH can predict clinically significant hypoperfusive outcomes in a homogenous cohort of generally anesthetised patients undergoing abdominal surgery, taking into account thresholds of MAP and their time durations. The data for this study come from a prospective cohort study in which patients who underwent abdominal surgery between 1 October 2018 and 15 July 2019 in the university hospital in Katowice were included in the analysis. We analysed perioperative data to assess how various IOH thresholds can predict hypoperfusive outcomes . 508 patients were included in the study. The total number of cases of clinically significant hypoperfusion was 38 (7.5%). We found that extending durations of low MAP, i.e., below 55 mmHg, 60 mmHg, 65 mmHg and 70 mmHg, were associated with the development of either AKI, MI or stroke. It was observed that for narrower and lower hypotension thresholds, the time required to induce complications is shorter. Patients who suffered from AKI/MI/Stroke experienced more episodes of any of the IOH definitions applied. Absolute IOH thresholds were superior to the relative definitions. For patients undergoing abdominal surgery, it is vital to prevent the extended durations of intraoperative mean arterial pressure below 70 mmHg. Finally, there appears to be no need to guide intraoperative haemodynamic therapy based on pre-induction values and, consequently, on relative drops of MAP. Over the years, there has been an increasing level of evidence linking intraoperative hypotension (IOH) to mortality, even one-year post-surgery . IOH cann = 11), reoperations (n = 24), procedures performed under local anaesthesia or monitored anaesthetic supervision (n = 33), and those classified as immediate according to the NCEPOD Classification of Intervention ..19].p” value <0.05 was considered statistically significant. All variables with significance below 0.05 in univariable testing were selected for the stepwise multivariable logistic regressions.Statistical analysis was performed using MedCalc Statistical Software version 18.1 . Continuous variables were expressed using medians and interquartile ranges (IQR). Qualitative variables were expressed as absolute values and percentages. Between-group differences for quantitative variables were assessed using the Mann–Whitney U test or Kruskal–Wallis test. Their distribution was verified with Shapiro–Wilk test. Chi-square or Fisher’s exact test was applied for qualitative variables. Areas Under the Receiver Operating Characteristics (AUROC) curves with 95% CIs were calculated to assess the diagnostic accuracy of consecutive models. All tests were two-tailed. A “The study group comprised 508 patients: 239 (46.6%) males and 269 (53.4%) females. The median age of patients was 62 years (IQR 46–68). Detailed study group characteristics are presented in Depending on the threshold applied, IOH could be diagnosed in 10.7–92% of all patients. The number of IOH cases increased gradually with the restriction of the IOH threshold .The composite primary outcome was diagnosed in 38 (7.5%) patients, including 32 cases of AKI (6.3%), 3 cases of MI (0.6%) and one event of stroke (0.2%). The association between the occurrence of a certain IOH threshold and the outcome is presented in In regards to the number of episodes of certain IOH thresholds, a significant difference between patients who suffered from the hypoperfusion outcomes and patients who did not experience it was observed. With each increase in the IOH threshold, the number of episodes of such hypotension increased and was greater for patients who experienced hypoperfusive outcomes . By usinIn this fairly homogenous abdominal surgery cohort, we found that extending durations of low MAP, i.e., below 55 mmHg, 60 mmHg, 65 mmHg and 70 mmHg, were associated with the development of either AKI, MI or stroke. There was a relationship between stricter IOH thresholds and their shorter duration required to be associated with the postoperative complications. Patients who suffered from AKI/MI/stroke experienced more episodes of any of the IOH definitions applied. Absolute IOH thresholds were superior to the relative definitions.p = 0.012) [The results of our study are consistent with previous reports ,10. In a= 0.012) .By introducing relative IOH thresholds, we sought to explore whether relative thresholds are better suited for predicting hypoperfusive events. We found that these thresholds are not good enough to predict postoperative complications as they were not significant in multivariable models. According to the POQI statement, absolute thresholds appear to be as predictive of renal and cardiac injury as relative thresholds . In our As mentioned above, there was a relationship between stricter IOH thresholds and their shorter duration required to be associated with postoperative complications. Interestingly, significant IOH thresholds  presenteThe above-mentioned findings should be interpreted with caution due to a number of possible confounding factors. Firstly, a pre-induction MAP value was defined as “baseline” MAP. It is possible that such measurement does not represent a true “baseline” as it could be influenced by stress or premedication. However, it was demonstrated that BP measurements obtained on the day of the surgery were similar to those obtained via primary care . SecondlThe major portion of the composite outcome consisted of AKI (32 cases). This explains why chronic kidney disease was such a strong predictor of the composite outcome . TherefoIn our study, blood pressures after discharging the patient from the operating theatre were not studied. However, the role of the postoperative care period in the adverse outcomes observed cannot be underestimated. Another potential limitation is that the size of our cohort may be underpowered to detect certain associations. It is possible that, e.g., higher IOH thresholds are still associated with postoperative complications, albeit not shown directly by our study. Finally, this is an observational study, and no causal association between IOH and hypoperfusive outcomes can be demonstrated.For patients undergoing abdominal surgery, it is vital to prevent the extended durations of intraoperative mean arterial pressure below 70 mmHg. There is a strong interplay between the severity and duration of intraoperative hypotension. Finally, there appears to be no need to guide intraoperative haemodynamic therapy based on pre-induction values and, consequently, on relative drops of MAP.


Resting heart rate is an independent predictor of colorectal cancer (CRC) development and CRC-related mortality. However, little is known about the relationship between resting heart rate and colorectal adenoma development. We aimed to investigate this association in a population who underwent screening colonoscopy.Among 39,021 patients who underwent both electrocardiogram and screening colonoscopy during routine health examinations at the Seoul National University Bundang Hospital, Health Promotion Center, Korea from January 2014 to July 2019, 1,344 patients had advanced adenoma. We performed 1:1 propensity score (PS) matching to establish a control group that mitigated the confounding effects of age and sex. We performed multivariate logistic regression analyses to identify the independent risk factors of advanced adenoma development.p = 0.006).Resting heart rate was significantly higher in the advanced adenoma group than in the control group. The prevalence of advanced polyp increased across the quartiles of resting heart rate. Patients with higher resting heart rates were more likely to be older, smokers, and have increased blood pressure and DM and less likely to engage in active exercises than those with lower resting heart rates. Patients with higher resting heart rates had higher serum glucose, triglyceride, hemoglobin A1C, and insulin levels and lower high-density lipoprotein cholesterol levels. Patients with resting heart rate in the highest quartile (≥71 bpm) still showed significantly increased odds ratio (OR) of advanced adenoma development (OR: 1.379, 95% confidence interval: 1.099–1.731, High resting heart rate was a meaningful independent risk factor of advanced adenoma development. AdvanceResting heart rate has been suggested to be an important predictor of all-cause mortality, cardiovascular mortality, and cancer mortality –9. RestiA previous study reported a high resting heart rate associated with a significantly increased risk for advanced adenoma recurrence as detected by surveillance colonoscopy in CRC patients . HoweverWe analyzed 39,021 patients who underwent both ECG and screening colonoscopy during routine health examinations at the Seoul National University Bundang Hospital, Health Promotion Center, Korea from January 2014 to July 2019. We excised all polyps detected during colonoscopy, and all specimens were sent to the pathology department for histological evaluation. Colorectal advanced adenoma was defined as a colorectal polyp with a diameter of >1 cm, or presence of ≥3 adenomas per patient, and/or villous component, and/or severe dysplasia based on the pathology specimens. A total of 1,344 patients had advanced adenoma in the present study. The study population was composed of 1,016 men (75.6%) and 328 women (24.4%). We performed 1:1 propensity score (PS) matching to establish control group without colon polyps that mitigated the confounding effects of age and sex. The exclusion criteria were as follows: patients with (i) incomplete medical records, (ii) a history of familial polyposis syndrome or Lynch syndrome, and (iii) known inflammatory bowel disease (IBD). This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital, and the requirement for obtaining written informed consent from the patients was waived.2) and obese (BMI ≥25 kg/m2) [Resting heart rate was assessed by 12-lead ECG in a supine position after >10 min of sufficient rest in all patients at the time of screening colonoscopy. Blood pressure was measured in a sitting position using an upper arm cuff oscillometric blood pressure device. The patients were instructed to avoid eating, drinking alcohol, smoking, exercising, and bathing for 30 min before the measurement. Height and weight were measured by trained nurses. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. The BMI was categorized following the World Health Organization (WHO) Asia-Pacific classification as normal (BMI <25.0 kg/m5 kg/m2) . Data ont-test or one-way analysis of variance (ANOVA) tests were used to compare continuous variables. Chi-square tests were used for categorical variables, as appropriate. We conducted an univariable logistic regression analysis for all variables. The variables which achieved a p-value < 0.05 after univariable logistic regression analysis were exported to the multivariable logistic regression analysis. Multivariate logistic regression analyses were carried out to identify the independent risk factors of advanced adenoma development, with adjustment for various confounders, including age, sex, alcohol history, smoking history, family history of CRC, exercise, BMI, HTN, DM, dyslipidemia, coronary artery disease, peripheral artery disease, cerebrovascular disease, arrhythmia, systolic blood pressure, and diastolic blood pressure. All statistical analyses were performed using the Statistical Package for Social Sciences . The PS matching to establish control group without colon polyps that mitigated the confounding effects of age and were performed by R package (version 3.2.2). A p-value of <0.05 was considered statistically significant. All authors had access to the study data and reviewed and approved the final manuscript.The means and standard deviations or medians and ranges were calculated for all continuous variables, as appropriate. Categorical variables were expressed as proportions (%), and statistical analyses were performed to compare the variables between groups. Independent two-sample Screening colonoscopy was performed on 39,021 patients from January 2014 to July 2019 at the Seoul National University Bundang Hospital, Health Promotion Center, Korea. Of the t39,021 patients, 19,826 (50.8%) patients had colorectal polyps. Moreover, 1,344 (3.4%) patients had advanced adenomas.Table 1). The advanced adenoma group had a higher proportion of patients who were smoking, engaging in less physical activity, with a higher BMI of >25 kg/m2, and with HTN, DM, peripheral artery disease, and cerebrovascular disease. Regarding the metabolic parameters, serum glucose, TG, hemoglobin A1C, and insulin levels were higher, and serum HDL cholesterol levels were lower in the advanced adenoma group than in the control group.The baseline characteristics of the advanced adenoma patients (n = 1344) and PS-matched controls (n = 1344) were investigated (Table 2. Patients with higher resting heart rates were more likely to be younger (p trend = 0.020), to be smokers (p trend = 0.040), to engage in less active exercise (p trend = 0.001), to have higher systolic blood pressure (p trend = 0.027), to have higher diastolic blood pressure (p trend < 0.001), and to have DM (p trend < 0.001).The baseline characteristics of the study participants according to quartiles of the resting heart rate are shown in p trend< 0.001), TG (p trend < 0.001), HDL-C (p trend = 0.002), HbA1C (p trend < 0.001), and insulin levels (p trend = 0.002) were observed among the groups (Table 2). No significant difference was seen for total cholesterol and LDL-C levels.When the metabolic parameters among the groups are compared according to the resting heart rate, significant differences in serum glucose (p < 0.001). The prevalence of advanced polyp showed an increasing tendency across the quartiles of resting heart rate .Resting heart rate was significantly higher in the advanced adenoma group than in the control group . After adjusting for age, the patients with resting heart rate in the highest quartile showed a significantly increased OR of advanced adenoma development . After adjusting for multiple variables, the patients with resting heart rate in the highest quartile still showed a significantly increased OR of advanced adenoma development . Smoking history , high BMI of >25 kg/m2 , and history of DM , peripheral artery disease , and cerebrovascular disease  were also positively associated with an increased risk of advanced adenoma development (Table 4). Active exercise was negatively associated with the development of advanced adenoma .In the univariate analysis, the patients with resting heart rate in the highest quartile (>70 bpm) showed significantly increased odds ratio (OR) of advanced adenoma development, compared to those with resting heart rate in the lowest quartile (≤58 bpm.) (OR: 1.488, 95% CI: 1.202–1.842, Resting heart rate is known as an independent risk factor for all-cause mortality, cardiovascular mortality, cancer mortality, and CRC-related mortality –10. RestOne possible mechanism explaining the decreased risk for advanced adenoma development by active exercise might be the mediating effect of the resting heart rate. Active exercise could increase in resting parasympathetic tone, decrease in response to beta-adrenergic stimulation, and contribute to a decrease in resting heart rates . The mosThe patients with high resting heart rates were more likely to be smokers. Linneberg et al. demonstrated that smoking heaviness has a causal relationship with a high resting heart rate . SmokingThe resting heart rate is significantly associated with the presence of metabolic syndrome . DM, insThe resting heart rate could be corrected through lifestyle modification, including engaging in active exercises and cessation of smoking, which could eventually improve the metabolic parameters. Many previous studies investigated the effect of beta blockers, because of the weakened norepinephrine signaling in CRC patients. Some studies reported the protective effects of beta blockers in CRC patients –29 Beta This study has several limitations. First, we are unable to explain the causal relationship between resting heart rate and advanced adenoma development because of the study’s retrospective, cross-sectional, and case-control design. Second, we only analyzed a single measurement of the resting heart rate and metabolic parameters; we did not perform repeated measurements of these parameters. Nevertheless, we obtained the accurate resting heart rate by 12-lead ECG in a supine position after >10 minutes of sufficient rest in all patients. Third, the measurement of the resting heart rate could be influenced by multiple factors. Fourth, the heart rate variability, linked to cancer prognosis and survival, was not investigated in this study ,37. FiftIn conclusion, the resting heart rate was positively associated with the development of advanced adenoma in patients who underwent screening colonoscopy during routine health examinations. Patients with high resting heart rates were more likely to be smokers, to be engaging in less active exercises, and to have DM. We consider that healthy lifestyle modifications aimed at controlling the resting heart rate could decrease the risk of developing advanced adenoma.


The prevalence of diabetes reaches epidemic proportions. Diabetes is the leading cause of end-stage kidney disease (ESKD) since 30–40% of diabetic patients develop diabetic nephropathy. Albuminuria and glomerular filtration rate used to assess kidney function are considered surrogate outcomes of chronic kidney disease. The search for a biomarker that predicts progression to diabetic kidney disease is intense. We analyzed the association of serum advanced glycation end-products (AGEs) index (AGI) with impaired kidney function in poorly controlled type II diabetic patients. We observed an association between AGI and impaired kidney function in microalbuminuria patients with hyperglycemia. A significant association between AGEs, particularly carboxymethyl lysine (CML), and impaired kidney function were observed. Administration of AGEs to mice showed heavy proteinuria and glomerular abnormalities. Reduced podocyte number in mice administered with AGEs could be attributed to the epithelial-mesenchymal transition of podocytes. Our study suggests CML could be independently related to the podocyte injury and the risk of DN progression to ESKD in patients with microalbuminuria. AGEs in general or CML could be considered a prognostic marker to assess diabetic kidney disease. Advanced glycation end-products (AGEs) index is associated with proteinuria among diabetic subjects.Carboxymethyl lysine, a well-characterized AGE, correlated with podocyte injury.Administration of AGEs to mice manifested in EMT of podocytes and impaired kidney function.Inhibitor against receptor for AGEs prevented podocyte injury and improved proteinuria.Diabetes has long been a growing epidemic, and Asia accounts for 60% of the world's diabetic population , 2. The Biomarkers play an essential role in the early detection of DN and its progression to ESKD, whereas microalbuminuria is one of the predominant markers . Microalin vivo formation of AGEs  has proven to be a reliable prognostic marker in the general diabetic population, it may not be valid in patients with diabetes and chronic kidney disease . It is dThe primary antibodies are as follows: anti-E-cadherin (#3195), anti-N-cadherin (#13116), anti-col IV (#PAA180Hu01), and anti-fibronectin (#PAA037Hu01) were purchased from Cloud-clone . Anti-nephrin (#NBP1-77303) and anti-podocin (#JB51-33) were purchased from Novus Biologicals . Anti-α-SMA (#ab5694) was purchased from Abcam . We obtained glutaraldehyde solution (#G5882) and other chemicals from Sigma-Aldrich .Study subjects were enrolled from outpatients attending diabetes specialties centers in Vijayawada and Guntur in the state of Andhra Pradesh, India. We recruited 130 subjects with albuminuria ranging from 150 to 450 mg/day. Inclusion criteria are diabetes with more than 5 years, persistently inadequate glycemic control, and proteinuria above 150 mg/day. These subjects are devoid of other diabetic complications such as diabetic retinopathy, diabetic neuropathy, and cardiovascular complications at recruitment. Exclusion criteria were hematuria, clinical and laboratory findings suggestive of non-diabetic glomerulopathy, and secondary kidney damage due to hypertension. Informed consent was obtained from all individual participants included in the study. This study was carried out following “The Code of Ethics of the World Medical Association (Declaration of Helsinki)” and approved by the Institutional Review Board of Guntur General Hospital, Guntur, Andhra Pradesh, India (GMC/IEC/120/2018).2). Blood pressure (BP) was monitored thrice by a digital oscillometer . Fasting blood glucose (FBG) was estimated in the whole blood using a glucometer . Blood samples  were collected in heparin tubes and were centrifuged at 3,500 rpm, 4°C for 20 min to separate plasma and RBC. HbA1c was estimated in whole blood using a D-10 analyzer (Bio-Rad#12010405) based on the principle of fully automated boronate affinity assay. We collected 24 h urine and early morning spot urine, and albumin content was determined by kit from BioSystems .Anthropometric measurements, including weight, height, and waist measurements were recorded for the patients. Body mass index (BMI) was calculated using the formula: weight (kg)/ height  were collected from the pathology lab. Patients who underwent nephrectomy for a localized kidney tumor were selected for the control group (n = 30). The non-affected part of the kidney tissue was utilized for histological examinations. Clinical information of these patients was obtained through the medical records available at intensive care units where patients were hospitalized.Archived kidney biopsies of DN patients , periodic acid–Schiff (PAS) stain, and Masson's trichrome stain. Kidney specimens were scored by two pathologists blinded each other and to the patients' identification and clinical data. Kidney injury score was assessed according to Tervaert et al. . At leasGlucose-derived AGEs were prepared as reported earlier . BrieflyHuman podocytes were maintained and differentiated essentially as detailed earlier . Differen = 6, each group). FPS-ZM1 is an inhibitor for a receptor for AGEs (RAGE). Mice in the control group received an equal volume of phosphate buffer as a vehicle, whereas the experimental group received i.p. injections of in vitro prepared AGEs (10 mg/kg b.w); AGEs, and FPS-ZM1 (1 mg/kg b.w) on a daily basis for 4 weeks. At the end of the experimental period, 24 h urine was collected to measure GFR, albumin, and creatinine levels. Additionally, urine was subjected to SDS-PAGE and stained with silver nitrate to visualize the proteins in urine. Animals were perfused, and kidneys were harvested. Kidney sections from paraffin-embedded tissues were used for immunostaining, and the glomerular lysate was used for immunoblotting and mRNA expression analysis.The Animal experimental procedures were performed in adherence with the Institutional Animal Ethics Committee of the University of Hyderabad. C57BL/6J male mice  were used in this study. These mice were randomly distributed into three groups viz. Control, AGEs, and AGEs+FPS-ZM1 , and AGI were significantly different between the controls and diabetic patients  and type II diabetic patients are provided in patients . TogethePoor glycemic control in diabetics is presented with excess AGEs . Therefowww.Nephroseq.org, University of Michigan, O'Brien Renal Center) also corroborated with our observation that in DN, decreased expression of epithelial marker (E-CAD/CDH1) and increased expression of mesenchymal marker (N-cadherin/CDH2) and transcription factors that elicit EMT phenomenon such as SNAI1 and TWST1  inhibitor protected the mice from podocyte EMT and glomerulosclerosis  associated with diabetic cataracts (AGI might represent a better glycemic control marker than HbA1c in diabetic patients with kidney insufficiency. In a recent study, Chitra et al. reported that plasma AGI was significantly (ataracts . In a reataracts . SimilarThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Guntur Medical College, Guntur General Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by University of Hyderabad.RN, VT, KP, and AP planned and designed the study. RN, VT, and KP collected patients' data. RN, RK, MM, and AS performed the experiments. RN and AP evaluated the data. RN, VT, and MM performed the statistical analysis. RN and AP wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The neutrophil-lymphocyte ratio (NLR) is easily calculated blood test parameter, which can be used as marker to predict many inflammatory disorders. The aim of this study was to assess and compare the NLR in maternal blood with the white blood cell (WBC) count and C-reactive protein (CRP) concentration for the prediction of histological chorioamnionitis.+ 0 and 34+ 6 weeks. Blood samples, collected less than 48 h before delivery and at least 48 h after the administration of corticosteroids, were selected for the analysis. The NLR was calculated by dividing the number of neutrophils by the number of lymphocytes. Chorioamnionitis was diagnosed by the histopathological evaluation of placental membranes and chorionic plate.This was a case-control study of 137 woman with preterm premature rupture of membranes (PPROM) at a gestational age between 22Patients with diagnosed histological chorioamnionitis (HCA) had significantly higher levels of WBC, CRP and NLR . Levels of WBC, CRP and NLR predicted HCA with an area under the curve (AUC) of 0.81, 0.81 and 0.89, respectively. NLR had statistically significantly higher AUC than WBC, but no significant difference was found between AUCs of NLR and CRP. The cut-off level of NLR was found to be 5,97, which had a sensitivity of 77 % and a specificity of 95 %.NLR has a good predictive value for HCA and could be used as an additional diagnostic marker for predicting histological chorioamnionitis in cases with preterm premature rupture of membranes before 34 weeks of gestation. Preterm premature rupture of the membranes (PPROM) is the spontaneous rupture of foetal membranes during pregnancy prior to 37 weeks of gestation. One of the most serious complications of PPROM is chorioamnionitis, which complicates almost half of all PPROM cases  and is aThe white blood cells (WBC) and C-reactive protein (CRP) are inflammatory markers used worldwide for the early diagnosis of chorioamnionitis. A systemic review and meta-analysis performed by Sabogal with colleagues demonstrated the low sensitivity 51 %) and specificity 65 %) of leucocytosis for the diagnosis of histologic chorioamnionitis (HCA) [1 % and s5 % of leThe neutrophil–lymphocyte ratio (NLR) is a marker of systemic inflammation that is calculated by dividing the absolute neutrophilic count by the absolute lymphocyte count . The NLRThe aim of this study was to assess and compare the NLR in maternal blood with the WBC and CRP concentration to determine the best maternal blood inflammatory marker for predicting HCA.This case–control study included 185 patients who were diagnosed with PPROM prior to 34 weeks of gestation and were admitted to Vilnius University Hospital Santaros Klinikos between July 2017 and July 2019. The study was approved by the Vilnius Regional Biomedical Research Ethics Committee (2017-07-04 No. 158200-17-931-434), and all participants signed an informed consent form before enrolment.+ 0–33+ 6 weeks; (4) diagnosed with PPROM; and (5) the absence of a maternal hypertensive disorder, gestation diabetes and intrahepatic cholestasis. Exclusion criteria included (1) multiple gestations; and (2) foetal malformations.The inclusion criteria were as follows: (1) maternal age ≥ 18 years; (2) singleton gestation; (3) gestational age 22Clinical characteristics such as maternal age, gravidity, parity, gestational age, newborn birthweight, Apgar score, and umbilical cord arterial pH were retrieved from the patients’ medical records.According to the institution’s standard protocol, all patients with diagnosed PPROM prior to 34 weeks of gestation were managed with antibiotics, a single course of antenatal corticosteroids, and tocolytics. Intravenous ampicillin 2 g and oral erythromycin 250 mg every 6 h were used for 48 h. The patients were then placed on oral amoxicillin 500 mg every 8 h and erythromycin 250 mg every 6 h to complete a 7-day course of antibiotic therapy. Two doses of 12 mg of dexamethasone given intramuscularly 12 h apart were used for accelerating fetal lung maturation. We analysed blood samples collected 24–48 h before delivery. Additionally, due to the presence of corticosteroid-induced leucocytosis, we only analysed blood samples that were collected at least 48 h after the administration of corticosteroids. Thus, 137 participants were included in the final analysis .According to the institution’s standard protocol, all postpartum placentas were examined histologically after preterm delivery. Classification of placental lesions was based on Amsterdam Placental Workshop Group criteria , gradingp-Values of < 0.05 were considered statistically significant for all tests.Data analysis was performed using R package (version 4.0.3) . The distribution of the data was determined by the Shapiro–Wilk test. All continuous variables were not normally distributed and are presented as the medians and interquartile ranges (IQRs). The categorical data are expressed as frequencies and percentages. Differences were compared between the two groups using the Mann–Whitney U test for continuous variables and the Pearson Chi-square test for categorical variables. Receiver operating characteristic (ROC) curves were constructed to estimate the ability of variables to differentiate between the groups. The area under the curve (AUC) was calculated to indicate the average sensitivity of a marker over the entire ROC curve. The DeLong test was used to compare the AUCs of the different models. The best cut-off values to predict the outcome were determined by the Youden index. A multivariate logistic regression analysis was performed to evaluate independent prognostic factors associated with HCA. p-value = 0.01). Also, the time from steroid administration to delivery was longer in Group I than in Group II .The study included 137 patients with preterm premature rupture of the membranes prior to 34 weeks of gestation: 52 patients in Group I and 85 patients in Group II. The clinical characteristics of the patients and their distribution within the groups are listed in Table The white blood cell (WBC), neutrophil and lymphocyte counts, C-reactive protein (CRP) concentration, and the NLR are shown in Table To assess the relative importance of blood inflammatory markers in the prediction of HCA, we performed a multiple logistic regression analysis. CRP and NLR were found to be the independent variables and were significantly associated with the occurrence of HCA Table .Table 3p-value = 0.08). Additionally, we constructed a ROC curve of the WBC and compared it with the ROC curve of the NLR. The results were approaching an acceptable significance level . Moreover, we assessed the effectiveness of CRP as an additional diagnostic marker to NLR for predicting the occurrence of HCA. This model predicted the occurrence of HCA with an AUC of 0.9 but did not differ significantly from the AUC of NLR alone .ROC curves were constructed to determine the ability of CRP, WBC and NLR to differentiate between groups. Figure 9/L for WBC and 5.97 for NLR. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the markers are demonstrated in Table The optimum cut-off values for CRP, WBC and NLR were identified using the Youden index. Regarding the prediction of histological chorioamnionitis, the cut-off value was found to be 8.5 mg/L for CRP, 12.62 × 10The NLR has been proposed as an additional infection marker and a potential parameter for predicting bacterial infection. The NLR increases following the progression of inflammatory disease: chemokines release neutrophils from the bone marrow and increase their life span in the blood , 16, whiOur study demonstrates that an increased NLR is an appropriate indicator for the prenatal diagnosis of HCA. According to the ROC curve analysis, the prognostic value of NLR does not differ significantly from that of the CRP, but it has greater prognostic value than the WBC and may be used as additional marker to predict HCA. The optimal NLR cut-off value to predict HCA was found to be 5.97 with a sensitivity of 77 % and a specificity of 95 %.To our knowledge, other published studies on the use of the NLR in the prediction of chorioamnionitis have been sporadic , 19. KimRecent studies focused on the ability of the NLR to predict preterm delivery. Kim and colleagues found that a combined model consisting of cervical length and NLR had a higher predictive value for spontaneous preterm delivery than using cervical length alone or other inflammatory markers such as CRP and WBC . A studyPatients with an imminent preterm delivery prior to 34 weeks of gestation receive corticosteroid therapy to accelerate foetal lung development. Corticosteroids are known to increase the WBC and predominantly the neutrophil count. The biological effects that contribute to the increase in neutrophils are the release of immature neutrophils from the bone marrow into the circulation, the demargination of neutrophils from the endovascular lining, the delayed migration of neutrophils into tissue, and the lower rate of apoptosis –24. LeucThe NLR changes during some pregnancy-related disorders. The NLR may increase in preeclampsia, gestational diabetes mellitus, and intrahepatic cholestasis –28. FurtOur clinical expectation is that the NLR together with other maternal blood inflammatory markers will improve the prediction of histological chorioamnionitis. NLR analysis is simple, inexpensive, and easily obtained.To the best of our knowledge, this is the first study to evaluate the NLR for the prediction of HCA in patients with PPROM prior to 34 weeks of gestation. Additionally, this study excluded blood samples affected by the administration of corticosteroids and cases with preeclampsia, gestational diabetes mellitus, and intrahepatic cholestasis. The limitation of our study is its retrospective design. Further prospective cohort studies are required to evaluate our result and to produce stronger evidence. Moreover, chorioamnionitis was defined by histological examination of the placenta. Although histological chorioamnionitis is the gold standard for diagnosing intrauterine infections , there iThe NLR has a good predictive value for the occurrence of HCA and could be used as an additional diagnostic marker for predicting histological chorioamnionitis in cases with preterm premature rupture of the membranes prior to 34 weeks of gestation.