Introduction. Low birth weight is an important indicator of the health of babies. A low birth weight is a leading health problem and a major reason for death in newborns. This study targeted to assess the relationship between maternal and infant factors and low birth weight in Iran through a systematic review and meta-analysis.Materials and Methods. This paper was a systematic review and meta-analysis of the relationship between maternal/ infant factors and low birth weight based on the published research papers conducted in Iran. To achieve this goal, two trained researchers independently elicited all the relevant articles by using the appropriate keywords and their combinations in SID, Madlib, Iranmedex, Irandoc, Google Scholar, Pubmed, ISI, Scopus and Magiran databases. The results of the study were combined with SPSS 20 and STATA software.Results. In the initial stage, 25 more relevant articles out of 46 papers were selected. The gestational age with less than 37 weeks and prenatal care had the most  and the least  effect on the low birth weight in newborns, respectively.Conclusion. This study showed that there is a significant relationship between the low birth weight and multiple births, pre-eclampsia, maternal weight gaining during pregnancy, baby’s gender, and pregnancy age. Hence, controlling the factors above in mothers during pregnancy by the health authorities could lead to the birth of infants with a healthy weight and consequently the number of infants with low birth weight will decrease. By low birth weight, we mean a birth weight of less than 2500 grand and by very low birth weight, we mean a birth weight of less than 1500 gr. Additionally, an immensely low birth weight is used for those infants with less than 1000 gr birth weight [2]. Due to their exceptional circumstances, infants with a low birth weight are not capable of adapting themselves outside the womb and are at a greater risk of mortality [3]. The mortality of infants with low and very low birth weight is respectively of 40 and 200 times more than that of the normal-weight newborns. In Iran, two-thirds of the infant deaths in the first 24 hours after birth, occur in infants with a low birth weight [4].Nowadays, low birth weight is one of the most severe health problems all around the globe [5]. In Iran, this statistics is equal to 11.56 % [6]. A low birth weight in both advanced and developing countries imposes a tremendous pressure on the health care system and family members together [7]. Many factors affect the low birth weight, among others, genetic, environmental, embryonic, placental and maternal could be mentioned . Also, the socioeconomic status, maternal age, ethnic factors, occupation, educational background of the mother and birth season seem to be influential in birth weight [9-12]. Based on several studies, the risk of low birth weight in mothers under 19 and over 35 has been confirmed [13]. Women over thirty-five years old are at high risk of maternal and fetal complications including obesity, chronic hypertension caused by pregnancy, etc., prenatal complications including preterm birth, stillbirth, low birth weight, intrauterine growth retardation, admission in the neonatal intensive care unit and congenital malformations [14]. The prevalence of a low birth weight varies in different populations. According to the reports published by the World Health Organization (WHO) in 2000, totally 18 million children are born with a low birth weight per year, which is equal to 14% of all the births . All the items and dissertations relevant to low birth weight were studied until the end of the year 2014. The researches lacking sufficient information or those inaccessible were excluded from the study. To avoid bias, two well-trained researchers searched the articles independently. Based on this strategy, 46 relevant articles were found, among which four repetitive and 12 irrelevant items, which were excluded from the study. After reviewing the abstracts, five pieces lacked required information and thus were dropped. Finally, 25 articles met the required criteria and were included in the meta-analysis step  and those that gained the required score were included in the survey.The exclusion criteriaAfter evaluation, the studies that did not gain a favorable rating, those conducted on a particular group or those that lacked a sufficient number of samples or samples of other diseases associated with the risk factors, were excluded from the study.Statistical analysisThe statistical analysis of the data was done through the random pooled relative risk (effect method) and Review manager 4.2 software. In order to assess the amount of disparities among the results, I2 factor was used and for analyzing the data, SPSS20 and STATA software were also used. Table 1.In this study, 46 articles based on searching in different databases were selected among which four duplicate and 12 irrelevant articles were excluded from the study. Finally, after reviewing the full text of 30 articles, 25 articles were included in the survey. The information regarding the selected items is presented in The variables tested in this study included the relationship between preterm birth, infant gender, maternal weight gain during pregnancy, the mother’s prolificacy, infection due to preeclampsia and health care during pregnancy.Fig. 2). The dispersion test was also positive for the calculation (I286.9%: and P < 0/ 0001).The analysis of 12 studies that investigated the relationship between preterm birth and low birth weight was carried out in the country. The estimated odds ratio (OR) of LBW in women who had a preterm birth less of than 37 weeks, was 19.81 , which indicated that the preterm birth could lead to an increase of low birth weight , which statistically showed a significant relationship (I 2:56.7% and P = 0/ 002).The Odds ratio of premature birth of less than 37 weeks with a low birth weight of a 95% confidence interval in the studies reviewed was based on the date of publication and the author through random-effects model. The midpoint and the length of each segment indicated an Odds ratio and a 95% confidence interval in each case. The Diamond Mark showed the Odd ratio in all the studies, which indicated the relationship between the infant’s gender and the low birth weight. 18 studies conducted in Iran reviewed the odd ratio (OR) of low birth weight with female to male proportion, being estimated at 1.26  . The dispersion test was also positive (I 2: 89.5% and P < 0.0001).The risk of infants with a low birth weight in mothers who did not have an ideal weight during their pregnancy was estimated OR: 3.13  . The dispersion test for this calculation was I 2: 67.8% and P < 0.008.As far as the relationship between multiple birth and low weight, six articles were found in which the Odds ratio (OR) of infants with a low birth weight in multiple births was estimated at 16.68  .Based on six articles respecting the infection of mother to pre-eclampsia with a low birth weight, the Odds ratio of underweight infants birth in mothers infected to pre-eclampsia during their pregnancy was estimated at 5.99 . The scattering test for these calculations was I 2: 82.6% and P < 0.0001, which showed a significant relationship .By reviewing the relationship between the birth of underweight infants and prenatal care during pregnancy in mothers who had an inadequate care during pregnancy (less than six times), the following values were estimated OR: 1.15 , that did not show a significant correlation between them (The odds ratio of the inadequacy of caring during pregnancy and infants with a low birth weight of a 95% confidence interval in the studies was reviewed based on the date of publication and the author through the random-effects model. The midpoint and the length of each segment indicated the Odds ratio and a 95% confidence interval in each case. The Diamond Mark showed the Odds ratio in all the studies.In this meta-analysis study, 25 relevant articles including 11 case-control, 3 cohorts, and 11 cross-sectional studies were reviewed. In the assessment of the relationship between premature birth, 12 studies were perused. In order to study the infant’s gender, multiple births, mother infection to pre-eclampsia, caring during pregnancy, 18, 6, 6, nine articles were analyzed, respectively. Additionally, for the examination of the relationship between weight gain during pregnancy and low birth weight, totally six articles were reviewed. 27]. In Ranjiran’s study done in Markazi province, 44.7 of the pre-term infants and 3.1 percent of the term infants suffered a low birth weight [17], which showed a significant difference, this fact supporting our findings in this study as well. This study revealed that there was a strong correlation between premature birth and low birth weight, so that, infants of the mothers, who had a preterm birth, suffered a low birth weight of 19.8 times more than those children who were born at 37 weeks. In the study performed by Bahrami et al., 57.2 of the infants born through premature birth and only 2.6 of the children born through term birth were underweight [41], and it was also discovered that the mean of birth weight in male infants was more in comparison with the one of female children .In analyzing the relationship between the infant’s genders, the low birth weight was more common amongst female than male children. In a study conducted in Japan, a significant association among girls and low birth weight was observed , that being, the mean number of infants in mothers with proper weight gain was more than of those with an improper weight gain .This research showed that the mother’s improper weight gain during pregnancy might lead to an increase in the number of underweight infants, so that the number of underweight babies in mothers who gained 6 kg during pregnancy was 3.3 times more than those parents who acquired a proper weight during pregnancy. In the conducted researches, a significant relationship was found between the mother’s weight gain and the low birth weight [LimitationsSome of the limitations that we faced with during the study included the lack of access to the full text of some of the studies, sufficient information in the studies, access to some unpublished manuscripts and insufficiency of samples in some of the studies.Concluding remarksThe present paper was a systematic review and meta-analysis study that assessed the relationship between maternal and infant factors with a low birth weight in Iran. This study revealed that maternal factors including multiple delivery and premature birth have a great impact on the birth of such infants. There was a significant relationship between the gaining of a proper weight in mothers and the infection of mother to pre-eclampsia. Based on the finding of the study, health and care authorities are recommended to train parents in the respective fields and adopt the appropriate ways to protect mothers against the risk of a low birth weight.AcknowledgmentsThe authors would like to thank the Research Center and the Student Research Committee of Medical Science University of Ilam for their help in conducting this research.


The birth weight reference curve to estimate the newborns at risk in need of assessment and monitoring has been established. The previous reference curves from Indonesia, approximately 8 years ago, were based on the data collected from teaching hospitals only with limited gestational ages. The aims of the study were to update the reference curves for birth weight, supine length and head circumference for Indonesia, and to compare birth weight curves of boys and girls, first child and later children, and the ones in the previous studies.Data were extracted from the Maternal-Perinatal database between 1998–2007. Only live singletons with recorded gestational ages of 26 to 42 weeks and the exact time of admission to the neonatal facilities delivered or referred within 24 h of age to Sardjito Hospital, five district hospitals and five health centers in Yogyakarta Special Territory were included. Newborns with severely ill conditions, congenital anomaly and chromosomal abnormality were excluded. Smoothening of the curves was accomplished using a third-order polynomial equation.Our study included 54,599 singleton live births. Growth curves were constructed for boys (53.3%) and girls (46.7%) for birth weight, supine length, and head circumference. At term, mean birth weight for each gestational age of boys was significantly higher than that of girls. While mean birth weight for each gestational age of first-born-children, on the other hand was significantly lower than that of later-born-children. The mean birth weight was lower than that of Lubchenco’s study. Compared with the previous Indonesian study by Alisyahbana, no differences were observed for the aterm infants, but lower mean birth weight was observed in preterm infants.Updated neonatal reference curves for birth weight, supine length and head circumference are important to classify high risk newborns in specific area and to identify newborns requiring attention. Size at birth reflects fetal growth and health as well as provides important information on the newborns infant. Many studies have been carried out to construct a theoretical birth weight curve for gestational age , 2. The The prevalence of high risk newborns depends on the birth curve used . TherefoToday clinicians in most developing countries are using the Lubchenco’s reference curve for newborns , 25. HowIn 1992 the Maternal-Perinatal (MP) team was established in Yogyakarta with the aim of conducting MP audits and creating an MP database in the district hospitals including data collection on birth weight, supine length and head circumference of newborns. The aims of this study were to update the reference curves for birth weight, supine length and head circumference for Yogyakarta, Indonesia and to compare birth weight curves of boys and girls, first child and later children, and the ones in the previous studies.The study was conducted in Yogyakarta Special Territory (YST) whose population is made up of various ethnics in Indonesia. Nevertheless it has not represented the population of Indonesia as a whole. YST consist of five districts. Each district is served by a district hospital and a couple of health centers, of which only one was equipped for deliveries, and the referral hospital Sardjito. During the study period January 1, 1998 to December 31, 2007 all deliveries at Sardjito Hospital, the five district hospitals, and the five health centers equipped for deliveries were recorded. Approximately, 80% of the newborns in YST were delivered by trained health personnel, 65% of whom were delivered in Sardjito Hospital, five district hospitals and five health centers; the remaining 35% was delivered in private hospitals, maternity clinics, midwife clinics or at home by midwives .Our study population consisted of all newborns delivered at Sardjito Hospital, five district hospitals, five health centers and those referred from other health facilities within 24 h of birth.Lubchenco , 25, NikThe study was conducted by MP team based on MP database. The MP database in the district hospitals is part of MP audit, which is a district-based audit of maternal and perinatal mortality. The MP audit was introduced in Indonesia as a tool for continuous surveillance of the maternal-perinatal mortality and quality assurance of the obstetric and perinatal services into the domain of district health system , 29.The MP database was run in every district hospital by filling in the MP form daily. The data were validated monthly by the local team before they were sent to the MP center at the beginning of the next month and were computerized by a trained secretary. The data generation process from data collection, field editing, data form submission to the data center, and to data entry were continuously monitored to identify errors and logical inconsistencies.In Indonesia, primary health care services are conducted in health centers. The district hospitals are secondary health facilities that provide referral services in that area. Tertiary health facilities are made available at teaching hospitals, which are usually found in the capital of a province. However, for provinces without a teaching hospital, the services are provided by the provincial hospital, a government hospital in the capital of the province.The forms from the five district hospitals in YST were submitted to the MP center at Sardjito Hospital until 2001, meanwhile the MP team in the center checked and entered the data. However, from 2002 onwards all facilities were checked and they entered the data by themselves. Therefore the 1998–2001 data were available in the MP center while the 2002–2007 data were available in the health facilities. Unfortunately, an earth-quake struck the area in May 2006 and damaged the soft copy in computers, thus causing most of the data to be re-entered from the MP forms.The MP database contained information from the mother’s delivery to the neonatal period for each individual in the maternity and newborns facilities in YST. The newborns were followed up until they were discharged from the facilities. Trained health personnel filled in the MP forms. They contained information on identity, characteristics of the mothers, their pregnancy and delivery, and the newborns.Only live singletons with recorded gestational ages between 26 to 42 weeks and the exact time of admission to the neonatal facility were included in the study; meanwhile those with severely ill conditions , major congenital anomaly, and those admitted >24 h of age were excluded.In most developing countries, women especially in rural areas are unaware of the exact date of their last menstrual period (LMP). Thus, they could not calculate the expected date of delivery using the first date of the last menstrual period. Dubowitz  developeBirth weight, supine length, and head circumference were measured immediately after delivery. All infants were weighed to the nearest 10 g on a balance scale (readjusted using standardized weight as part of routine care). The length was measured using a measuring board with supports for the head and feet to the nearest cm. The head circumference was recorded using a measuring tape to the nearest cm. Training and standardization in anthropometric measurements of weight, length, head circumference, and clinical assessment of gestational age by Dubowitz score were carried out in December 1997. All measurements were examined by trained nurses.th, 5th, 10th, 25th, 50th (median), 75th, 90th, 95th, 97th percentiles by gestational age relevant for clinicians in classifying newborns under their care and to researchers as well as public policy makers in comparison to geographic differences and temporal trends in birth weight for gestational ages in population. All analyses were performed separately for boys and girls. Distribution of birth weight, supine length, head circumference at the corrected gestational ages was smoothened by a third degree polynomial function. Curves were produced using Microsoft Excel 2010.Data analysis was performed using SPSS version 19. Tables and graphs presented means and standard deviations (SDs) and the 3t-test. In the birth order of children, the term “first” refers to the 1st child, and “later” refers to second child and so on. The weight-length ratio was calculated according to Rohrer’s Ponderal index (PI); 100 x weight in grams/length [Difference in mean birth weight between boys and girls, as well as first and later-born for each gestational age was analyzed using Student’s s/length  in centis/length . The PI From January 1998 to December 2007 there were 59,609 births. Most of the infants (83.2%) were born in Sardjito Hospital, five district hospitals, and five health centers, whereas the others (16.8%) were born in other hospitals, health centers, midwife clinics, at home, and were admitted to the study setting before 24 h. In this study there were 54,599 subjects in total. Mean birth weight was 2,964 g and there was no difference in birth weight over time.Sardjito Hospital, the five district hospitals, and the five health centers in YST contributed with 25%, 56% and 19% of the newborns respectively. First child constituted 26,189 48.0%) and later child was 28,410 (52.0%). The numbers of eligible infants for birth weight, length and head circumference were 54,599, 52,261 and 48,109 respectively (53.3% boys and 46.7% girls)  mean birth weight for each gestational age was significantly higher for boys than for girls Table , Fig. 7 For gestational age ≥39 weeks there was a striking similarity in mean birth weight among Lubchenco’s, Alisyahbana’s, and our study. The mean birth weight for gestational age ≤38 weeks was lower in our study than that in Lubchenco’s. Gestational age 34–37 weeks presented the highest mean birth weight in Alisyahbana’s but the lowest in our study (Table Tertiles of PI of our study were low (<2.5), average 2.5–2.8) and high (>2.8). The PI of term boys, girls, first and later children in our study were classified into average group. In the preterm, however, it was classified into low group Tables  and 7. T and highOur study presented girls and boys for birth weight, length and head circumference based on the local data. One of the weaknesses of our study was that it did not have enough low-gestational age infants. Therefore the application of the curve in low gestational age infant must be done carefully.Moreover, comparison of each gestational age showed higher significance in at term only, but not in preterm. The result was similar to the study by Fok  whereby p < 0.001) than first-born children. In preterm there was no significant difference, though. Nevertheless, Alisyahbana reported that for every gestational age and percentile, later-born children were heavier than first born-children [Skjaerven  pointed children .We could not compare the mean birth weight for each gestational age in our study and that in the previous studies by Lubchencho and Alisyahbana, since there was no information on standard deviation. Thus, the comparison was based on mean birth weight for sexes combine because no information of separated boys and girls was found in Alisyahbana’s. Similarly, comparison of our study and Lubchenco’s showed that for gestational age ≤38 weeks the mean birth weight was lower in our study. This was probably due to the relatively high number of infants with small for gestational age in our population for term and preterm, which needed further investigation.Compared with Alisyahbana’s study, for gestational age 34–37 weeks the mean birth weight was lower in our study; which was probably due to the differences of sample. Our study had more data from health centers, district hospitals, and 1 teaching hospital, whereas Alisyahbana’s study collected the data from 14 teaching hospitals with middle and high socio-economic status. In addition, the numbers of samples in our study were much higher with updated reference for 26 to 42 weeks gestational age, meanwhile Alisyahbana’s was only 34–42 weeks. Unfortunately, we could not compare our result with Niklasson’s curve , since wTertiles of PI for our study were similar to those of Morris’s  report, Lubchenco  reportedThus, the combination of short and low PI at birth may well provide a useful classification of the anthropometric status of the newborns. Infants who were born short with low PI were at risk of mortality and severe morbidity during infancy . The lowrd and 97th percentiles, −2 SD or +2 SD were added. We expect that these curves would be useful for the care of Indonesian newborns.Important cut off points for risk assessment of the 3Our study separated girls and boys for birth weight, length and head circumference based on the local data. At term, mean birth weight of boys was significantly higher than that of girls, mean birth weight of first-born children was significantly lower than that of later born-children; but in preterm, both did not suggest significant difference.For gestational age ≥39 weeks there was mean birth weight similarity to Lubchenco’s, Alisyahbana’s, and our study. When compared with Lubchenco’s study, the mean birth weight for gestational age ≤38 weeks was lower in our study. However, for 34–37 weeks, the mean birth weight in our study was lower than that in Alisyahbana’s study.The PI of term for boys and girls and first and later-born children was classified into average, whereas that of preterm was classified into low. The PI for gestational age ≤35 weeks was lower in our study than in Lubchenco’s; however, for gestational age ≥39 weeks it was higher in our study than in Lubchenco’s and Alisyahbana’s.Updated and improved neonatal reference curves for birth weight, supine length, and head circumference are important to classify high risk newborns in specific area and to recognize those requiring attention with regard to recent condition.


The potential benefit of SGLT2 inhibitors in metabolic syndrome is with prediabetic stage unclear. This work was undertaken to investigate the non-glycemic effect of empagliflozin on metabolic syndrome rats with prediabetes.SHR/NDmcr-cp(+/+) rats (SHRcp), a model of metabolic syndrome with prediabetes, were given empagliflozin for 10 weeks to examine the effects on urinary sodium and water balance, visceral and subcutaneous adipocyte, and cardiac injury. Further, the effect of empagliflozin on blood pressure and autonomic nervous system was continuously investigated by using radiotelemetry system.Empagliflozin significantly reduced urinary sodium and water balance of SHRcp only within 1 week of the treatment, but later than 1 week did not alter them throughout the treatment. Empagliflozin significantly reduced body weight of SHRcp, which was mainly attributed to the significant reduction of subcutaneous fat mass. Empagliflozin significantly reduced the size of visceral adipocytes and increased the number of smaller size of adipocytes, which was associated with the attenuation of oxidative stress. Empagliflozin ameliorated cardiac hypertrophy and fibrosis of SHRcp, in association with the attenuation of cardiac oxidative stress and inflammation. However, empagliflozin did not significantly change blood pressure, heart rate, sympathetic activity, or baroreceptor function, as evidenced by radiotelemetry analysis.Our present work provided the evidence that SGLT2 inhibition reduced visceral adipocytes hypertrophy and ameliorated cardiac injury in prediabetic metabolic syndrome rat, independently of diuretic effect or blood pressure lowering effect. Thus, SGLT2 inhibition seems to be a promising therapeutic strategy for prediabetic metabolic syndrome.The online version of this article (doi:10.1186/s12933-016-0473-7) contains supplementary material, which is available to authorized users. Type 2 diabetes is a major risk factor for cardiovascular disease , 2. HoweAlthough SGLT2 inhibitors are shown to exert not only antihyperglycemic effect but also non-glycemic effects such as natriuresis, body weight reduction, blood pressure-lowering effect, or lipid-lowering effect, the previous reports regarding these non-glycemic effects of SGLT2 inhibitors are limited to diabetic animals –20 or paAll procedures were performed in accordance with the institutional guidelines for animal research and were approved by the Animal Care and Use Committee of Kumamoto University.Male SHR/NDmcr-cp(+/+) rats (SHRcp) –29, a raEmpagliflozin, a selective sodium-glucose cotransporter-2 (SGLT2) inhibitor, was kindly supplied by Boehringer Ingelheim Pharma GmbH & Co. .The present study consisted of three experiments as described below.This experiment was performed for metabolic cage analysis  and heart rate (HR) of SHRcp, using radiotelemetry system  /high frequency (HF) ratio of PI and LF of systolic BP) were calculated using data-analysis program contained a fast Fourier transform function for power spectral analysis established by Oosting et al. and Waki H et al. –33.+/g food). Twenty-four-hour sodium excretion was calculated by multiplying the 24-h urine volume (mL) and urinary sodium concentration (mmol Na+/mL). Twenty-four-hour sodium balance was calculated as 24-h sodium intake minus 24-h sodium excretion. Twenty-four-hour water balance was calculated as 24-h water intake minus 24-h urine volume. Cumulative sodium or water balance was calculated from sequential summation of daily balances according to our previous report [In the above mentioned Experiment I, 24-h sodium intake was calculated by multiplying food intake (g) by the diet sodium content . Positive fibrosis area per field area was assessed by examining at least 10 fields per rat using WinRoof Version 5.8 .2) was counted by examining more than ten fields per section using a microscope with 200× magnification; the average number of ED-1 positive cells was obtained for each rat.To evaluate cardiac interstitial macrophage infiltration, hearts were fixed with 4% formalin overnight, embedded in paraffin, cut into 4-μm thick coronal sections, and immunostained with anti-ED-1 antibodies  (working dilution 1:500) to identify monocytes/macrophages, as described previously . The numTo evaluate cardiomyocyte size , cardiacDihydroethidium (DHE) was used to evaluate tissue superoxide levels in situ, as described previously . DHE fluTriglyceride in left ventricular tissue was measured with a commercial assay kit  according to the manufacturer’s recommended protocol.To evaluate adipocyte size, epididymal  and subcutaneous adipose tissue were fixed with 4% formalin overnight, embedded in paraffin, cut into 4-μm thick coronal sections, and stained with haematoxylin and eosin. Adipocyte size was assessed by examining at least 100 cell sizes per rat using WinRoof Version 5.8 .Adiponectin protein levels in adipose tissues were analyzed using a mouse/rat adiponectin ELISA kit . Protein extraction from adipose tissue and protein quantification were performed as described by other investigators .Lipid oxidation was evaluated by measuring the amounts of malondialdehyde (MDA) produced thiobarbituric acid reactive substances (TBARS), using a commercial assay kit . Briefly, adipose tissue homogenate, sodium dodecyl sulphate, acetic acid and TBA were mixed. The mixtures were heated at 95 °C in a water bath for 60 min. After incubation the tubes were cooled to room temperature the upper organic layer was taken and its OD read at 532 nm against an appropriate blank without the sample.Serum total cholesterol, triglyceride, and free fatty acid, and urinary electrolytes were measured at SRL, Inc. . Measurement of HbA1C was performed at biopathological medicine . Plasma insulin was measured with a kit .All of the data are presented as mean ± SEM. The difference among comparison groups was tested with appropriate statistical method, as shown in each Figure legend and tables. P < 0.05 was considered significant. Statistical analyses were performed using GraphPad Prism version 6.0 for Windows .There was no significant difference in daily body weight and daily food intake between control and empagliflozin groups during 7 days of the treatment Fig. a, b, butBody weight began to be significantly less in empagliflozin group than in control group from 7 weeks after initiation of empagliflozin treatment Fig. a, while Different from the shot-term (7 days) effects of empagliflozin, there was no significant difference in 24-h urinary sodium excretion, sodium balance, and water balance between empagliflozin and control group until the end of the treatment 10 weeks) Fig. f, g, h. 0 weeks FAfter 10 weeks of the treatment, body weight was significantly less in empagliflozin group versus control group , while tibia length was similar between the groups  Table .Figure There is no significant difference between the control and empagliflozin groups regarding epididymal fat adiponectin (1.16 ± 0.07 versus 1.03 ± 0.08 ng/μg protein) and subcutaneous fat adiponectin (1.42 ± 0.09 versus 1.37 ± 0.08 ng/μg protein).As shown in Fig. As described above Table , left veAs shown in Table Blood pressure and heart rate were continuously monitored in SHRcp for 7 weeks by using telemetry system. As shown in Fig. Figure At present, available data regarding the effect of SGLT2 inhibition on MS with prediabetic stage is very scarce. The major findings of our present work were as follows: (1) empagliflozin significantly ameliorated cardiac hypertrophy and interstitial fibrosis in association with the attenuation of cardiac oxidative stress and inflammation in prediabetic MS rat, independently of blood pressure and lipid metabolism; (2) empagliflozin reduced visceral adipocyte hypertrophy in prediabetic MS rats. These results provided the first evidence suggesting that SGLT2 inhibition with empagliflozin may provide the therapeutic benefit in MS with prediabetic stage.Recently, EMPA-REG OUTCOME trial provides the evidence that SGLT2 inhibition with empagliflozin significantly reduces cardiovascular events and heart failure hospitalization in type 2 diabetic patients at high risk –14, 38. Clinical studies showed that empagliflozin monotherapy reduced postprandial glucose from the first day and improved 24-h glucose variability in Japanese patients with type 2 diabetes , the comConvincing evidence , 21, 26 The potential effect of SGLT2 inhibition on cardiac disease is paid the most attention, since empagliflozin is demonstrated to reduce cardiovascular events and heart failure hospitalization in type 2 diabetic patients –14, 38. Accumulating studies provide the solid evidence that long-term SGLT2 inhibition causes the reduction of body weight in type 2 diabetic patients , 24. HowIn conclusion, we first examined the detailed effect of SGLT2 inhibition on prediabetic MS rats. We obtained the evidence that empagliflozin ameliorated cardiac hypertrophy and fibrosis in prediabetic MS rats, through attenuation of oxidative stress and inflammation, independently of BP. Furthermore, empagliflozin treatment provided the reduction in visceral adipocyte hypertrophy and the attenuation of visceral adipocyte oxidative stress in prediabetic MS rats. Thus, our present work highlights SGLT2 inhibition as a novel therapeutic strategy for MS with prediabetic stage.


The pathogenesis of normal pressure hydrocephalus (NPH) remains unclear which limits both early diagnosis and prognostication. The responsiveness to intervention of differing, complex and concurrent injury patterns on imaging have not been well-characterized. We used diffusion tensor imaging (DTI) to explore the topography and reversibility of white matter injury in NPH pre- and early after shunting.Twenty-five participants  underwent DTI, pre-operatively and at two weeks post-intervention in patients. We interrogated 40 datasets to generate a full panel of DTI measures and corroborated findings with plots of isotropy (p) vs. anisotropy (q).p = 0·022) in the posterior limb of the internal capsule, compatible with improvement in stretch/ compression, that preceded any discernible changes in clinical outcome. On PQ plots, the trajectories of the posterior limb of the internal capsule and inferior longitudinal fasciculus suggested attempted ‘round trips’. i.e. return to normality.Concurrent examination of DTI measures revealed distinct profiles for NPH patients vs. controls. PQ plots demonstrated that patterns of injury occupied discrete white matter districts. DTI profiles for different white matter tracts showed changes consistent with i) predominant transependymal diffusion with stretch/ compression, ii) oedema with or without stretch/ compression and iii) predominant stretch/ compression. Findings were specific to individual tracts and dependent upon their proximity to the ventricles. At two weeks post-intervention, there was a 6·7% drop in axial diffusivity (DTI profiling with p:q correlation may offer a non-invasive biomarker of the characteristics of potentially reversible white matter injury. Cerebrovascular and neurodegenerative, including Alzheimer’s changes, are present in many NPH patients. Leptomeningeal fibrosis has been found but does not correlate with resistance to CSF outflow (Rcsf) . The undet al. found it possible to distinguish NPH from Alzheimer’s and Parkinson’s diseases as well as Lewy body dementia with a sensitivity of 86% and specificity of 96% [et al. demonstrated alterations in corticospinal tract microstructure sufficient to discriminate NPH from Alzheimer’s and Parkinson’s diseases, with a sensitivity of 94% and specificity of 80% [Various MR techniques have been applied to NPH including magnetic resonance elastography (MRE) . Diffusiy of 96% . Howevery of 96% , 13. Usiy of 80% . Early wy of 80% .We hypothesized that, in NPH, differing patterns of white matter injury could be demonstrated to occur concurrently. Our study aimed to demonstrate the effects of surgical intervention on pre-operative patterns of injury and to evaluate the responsiveness of individual DTI patterns to CSF shunting. We used a full panel of DTI measures to interrogate both the microstructural effects of NPH on key white matter tracts likely to be ‘at-risk’ of damage. Published guidelines recommend the earliest assessment of clinical outcome scoring to be at 3-months post-intervention . We examWe recruited sixteen patients with NPH  ranging in age from 60 to 84 years , learning and memory , verbal fluency and executive function (COWAT) –23. As e2-weighted sequence , a fluid attenuated inversion recovery (FLAIR) sequence, , a 3D volumetric T1-weighted (MPRAGE) acquisition . The DTI data set was acquired by using a spin echo diffusion-weighted echo planar imaging sequence with the following parameters: TR/TE, 8300ms/98ms, matrix dimensions 96x96, FOV 192x192, slice thickness 2mm giving a voxel size of 2x2x2mm. Diffusion weighted images were acquired in 12 non collinear directions each at 5 b-values of 350, 650, 1000, 1300 and 1600 s/mm2, along with 4 b-0 images.MRI was performed on a 3T Siemens Tim Trio using a 12 channel head matrix radio frequency receive coil. We performed imaging pre-operatively and at two weeks post-operatively. Healthy controls underwent one MR scan. The MR imaging protocol included a Tλ1, λ2 and λ3. λ1 was taken to represent diffusivity along the axonal fibres, i.e. axial diffusivity (λ║ or L1). The means of diffusivities perpendicular to the axonal fibres, λ2 and λ3, represented radial diffusivity (λ┴ or L2and3). Co-registration of the structural 3D volumetric image and DTI images was performed using SPM5  [0 volume .We analysed forty DTI datasets. We applied eddy current correction and DTI pre- and post-processing in the standard manner using the FDT diffusion toolbox in FSL analysis tools  –29. We gdon, UK) . We re-s3 spherical MarsBaR (3mm radius) to be the optimal ROI with maximal white matter and minimal CSF/ extra-axonal sampling. We believe such a refined target avoids the limitations associated with spatial normalization.Co-registration and reproducibility of the MarsBaR methodology from the SPM5 toolbox have been previously published from our unit . Prior tROIs were exported to Image J  and applied to all DTI output files . We visut-tests (two-tailed) were performed for patients compared to controls and paired-samples t-tests (two-sided) for pre-operative compared to post-operative patients. Results were deemed to be significant at a value of p < 0.05 on the Student’s t-test. We considered each white matter tract independently of each other; only two groups were compared in each experiment. Multivariate ANOVA-type analyses of DTI measures were not performed; ROI sets for each pairing were directly compared for the analysis of directional information and proportional magnitude of changes in order to generate DTI profiles; Bonferroni corrections were therefore not deemed appropriate in this study. We performed comparison of means and the percentage differences of changes in fractional anisotropy as well as mean, axial and radial diffusivities. By correlating significant findings with the simultaneous examination of the direction and magnitude of changes across the full panel of DTI measures, we determined predominant patterns of injury. Interpretation of DTI measures was determined a priori and derived from work published in human and animal studies . Independent-samples dies see . DTI patet al [et al [We have previously introduced p:q tensor transformation for the analysis of MR DTI images in Pena et al . This tel [et al , 36. OurThe first step in the p:q tensor transformation is to decompose the diffusion tensor D into two tensors P and Q according to the next equation:Here I is the identity tensorThe first term on the right hand side is the isotropic tensor (denoted tensor P), while the second term in brackets represents the deviatoric tensor (denoted tensor Q). The magnitude of these tensors can be quantified by its scalar isotropic (p) and anisotropic (q) components. The values of p and q can be computed as:Here D is the mean diffusion and the lambdas are the eigenvalues of the diffusion tensor. According to these definitions, p is therefore a scaled measure of the mean diffusion in the tensor, while q is a measure of the variance or deviation of the eigenvalues with respect to the mean diffusion of the tensor.The second step is to plot each tensor as a point in a Cartesian plane with p taken as the x-axis and q as the y-axis. This plane will be denoted as the p:q plane. The effect of this transformation is to reduce the dimensionality of the tensor from six dimensions to two.-4 mm2/s, RA and FA are dimensionless, and phi has units of degrees.The third step is to use the p:q plane to deduce the five additional tensor scalar measures: D, RA, FA, L and phi. Four of these seven tensor measures  are anisotropy measures, while D and p are measures of the magnitude of diffusion and L is a measure of the total diffusion of the tensor. D, p, q and L have units of x10These scalar measures can be deduced either analytically or graphically. The analytical method is to directly compute the measures based on the p, q components using the formulae:et al. have recently used it to distinguish intracranial epidermoids from normal white matter in the splenium of the corpus callosum [In previous work, we highlighted the potential pitfalls of using FA and RA as sole means of MR DTI analysis and the advantage of visually representing seven scalar measures simultaneously. The p:q tensor transformation has since been used in various clinical settings to quantify the diffusion and anisotropy in various tissues, thereby gaining information about their internal architecture. Srinivasan callosum . In thiscallosum , which gThe sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to all the data in the study and responsibility for the decision to submit for publication.p = 0.001; and body +26.1%, p = 0.002; p = 0.001 and -21.9%, p = 0.003 respectively; p = 0.002 and +48.3%, p = 0.002; p = 0.017 and +10.4%, p = 0.029; In NPH patients compared to controls, there was an increase in MD in all tracts with the greatest change in the corpus callosum  to result in a small increase, not a decrease, in FA  despite an increase in radial diffusivity .The inferior longitudinal fasciculus, anterior thalamic radiation and inferior fronto-occipital/ uncinate fasciculi demonstrated similar but less marked changes. In contrast, the increase in axial diffusivity in the posterior limb of the internal capsule was sufficiently large  between pre-operative NPH patients vs. healthy controls. Blue shading designates percentage differences (%) between post-operative NPH patients vs. healthy controls.Interpretation of pre-operative white matter injury patterns—i) Genu of the corpus callosum- the increase in radial diffusivity (L2and3) is > 4 times that of axial diffusivity (L1), although both the L1 and MD are increased. This is consistent with “predominant transependymal diffusion with stretch/compression”; profiles for the body of the corpus callosum were similar, ii) Inferior longitudinal fasciculus—there is an increase in L1 but this change is proportionally lower than the increases in MD and radial diffusivity . This is consistent with “stretch/oedema”; and iii) Posterior limb of the internal capsule—the increase in L1 is predominant  and there is no reduction of FA. This is consistent with “predominant stretch/compression”. Using the same methodology, the inferior fronto-occipital/uncinate fasciculi demonstrated “stretch/oedema’ but the anterior thalamic radiation had oedema without stretch (no increase in L1). The overlay of post-operative and pre-operative DTI profiles illustrate changes in NPH patients vs. healthy controls at two weeks post-intervention. Significant changes were demonstrated in the posterior limb of the internal capsule (FA and L1).p = 0.022) and FA  in the posterior limb of the internal capsule, together with a trend towards an increase in radial diffusivity . The post-operative FA was now less than healthy controls. The only other significant change was an increase in axial diffusivity in the body of the corpus callosum . On p:q examination, there were sharp decreases in anisotropy (q) in the genu of the corpus callosum, inferior longitudinal fasciculus and the posterior limb of the internal capsule  vs. anisotropy (q), that were not dependent upon prior knowledge of those profiles. When p, q coordinates were plotted in dimensionally comparable graphs, we found that differences in DTI profiles produced patterns of injury that occupied geographically distinct white matter districts . FurtherHow are DTI findings, consistent with measures described in our study, interpreted within the wider context of white matter injury? et al. originally reported findings of a generalised increase in brain ADC in five patients with NPH whereas Ivkovic et al. have suggested that MRI mean diffusivity histogram analysis is able to distinguish NPH from neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases with 86% sensitivity and 96% specificity [Our demonstration of increased mean diffusivity in all six tracts examined reflects increases in both axial and radial diffusivities and confirms earlier studies. Gideon cificity , 51. We per se may have no effect on neurological function unless it contains neurotoxic compounds [With normal ageing, there is a small increase in periventricular mean diffusivity . Increasompounds .It is clear that NPH is likely to be a balance of multiple injury elements co-existing. Neuroradiological findings have been mechanistically diverse, spanning changes from cerebral blood flow and CSF hydrodynamics to the architecture of structural compartments and gray matter volume . Whilst et al. suggested that, in children with acute hydrocephalus, stretching of the fibres to become more linearly aligned results in fewer obstacles to diffusion [et al. demonstrates their concept elegantly [stretch/ compression’. Whilst stretch remains the key concept, the combination term acknowledges the contradictory possibility of decreases vs. increases of radial diffusivity, such as seen in distortion around brain tumours vs. compression in fixed neural compartments. In the context of severe distortional stretch of fibres resulting in a larger component of increased radial diffusivity, we prefer the term ‘stretch/oedema’, which conveys the concept of leakiness across axons where stretch has been sufficient to result in compromised microstructure.One proposed mechanism with apparent merit is stretch. Assaf iffusion . Work inlegantly . This islegantly . Microstlegantly . Why shoIn imaging NPH, there is a difficult dichotomy between the apparent simplicity of features required for its diagnosis and the challenge of finding adjuncts proven to increase diagnostic accuracy of shunt-responsive NPH. Structural sequences are key to identifying significant ventriculomegaly, not entirely attributable to cerebral atrophy or congenital enlargement, and for confirming that there is no macroscopic obstruction to CSF flow . Howevera priori, in order to resolve such conflicts  that appeared to be influenced by neuroanatomical factors. We found that both the proximity of white matter tracts to the lateral ventricles and the directional arrangement of fibre tract bundles mattered. In general terms, tracts directly adjacent to the ventricles, such as the corpus callosum, were the most affected, with significantly abnormal findings across the entire panel of DTI measures. Tracts furthest away from the ventricles, arranged in a supero-inferior fashion, such as the posterior limb of the internal capsule, were the most preserved. Fibre tracts lateral to the ventricles, running antero-posterior, demonstrated a combination of the injury elements observed in the other two patterns.The most affected white matter tracts were the genu and body of the corpus callosum that are directly adjacent to the ventricles, consistent with other studies , 71. TheThe tracts lateral to but close to the ventricles, inferior longitudinal fasciculus, demonstrated similar but less severe changes consistent with stretch/compression and increased interstitial fluid. DTI findings in the inferior fronto-occipital/ uncinate fasciculi were also consistent with this profile, despite being further away from the ventricles. There is some, albeit limited, neuropathological evidence to support these interpretations , 72. TheBy contrast, the posterior limb of the internal capsule demonstrated the greatest increase in axial diffusivity and anisotropy , a modest increase in MD and radial diffusivity with no reduction in FA Figs  and 3. TAt two weeks following surgery, it was possible to demonstrate significant changes in axial diffusivity in the posterior limb of the internal capsule. There was also a modest increase in radial diffusivity, consistent with re-expansion of extracellular fluid spaces between axons. These changes are consistent with relaxation of stretch/compression following surgical intervention. The findings preceded changes in clinical outcome; whilst the significance of such results requires further clinical correlation, they nevertheless provide evidence of changes in DTI directly attributable to the effect of surgical intervention upon white matter injury patterns. Surgery did not impact upon all three pre-operative injury patterns in the same way; the pattern of predominant stretch/ compression was the most amenable to intervention. However, trends demonstrated such DTI changes following intervention could be seen for the profile of stretch/compression even if it was not predominant; changes were proportional to the magnitude of the component present pre-operatively. Conversely, other profiles progressed despite surgical intervention, implying that the absence of the profile of stretch/ compression may be disadvantageous, or indeed that other profiles of white matter injury are less reversible.a priori presumptions of predominant pathological injury. We found an increase in the isotropy of all white matter tracts as all ROIs evolved from healthy controls to pre-operative NPH patients, consistent with the interpretation of widespread axonal disruption or distortion  and diffusion tensor parameters, the post-operative changes seen in the genu of the corpus callosum, inferior longitudinal fasciculus and the posterior limb of the internal capsule were consistent with the theoretical prediction for decompression. Strikingly, changes in DTI and p:q profiles suggested that the body of the corpus callosum apparently worsened. We hypothesize that this may simply be a ‘shunt effect’. Changes in radial diffusivity and isotropy do not distinguish between inflow and outflow movements; in the early post-operative period, it is therefore not possible to distinguish between the continuing pathological process of transependymal diffusion vs. the increased flow into the ventricles due to the physiological process of CSF drainage due to shunting. This is in contrast to finite periods of continuous CSF diversion, such as external lumbar drainage, in which changes of decreased mean diffusivity could be more simply demonstrated [Two weeks post-surgical intervention, p:q changes for the genu of the corpus callosum, inferior longitudinal fasciculus and the posterior limb of the internal capsule suggested improvement in stretch/compression . Only twnstrated . The effThis study demonstrates the responsiveness of differing DTI profiles of white matter injury to surgical intervention. Demonstrating changes due to intervention does not necessarily equate to demonstrating clinical improvement secondary to shunting. In fact, as is quite commonly the case clinically, patients in this study were seen to merely return to baseline at this stage, following a period of post-operative functional recovery. It is for this reason that post-operative clinical outcome assessment, involving the use of NPH outcome scoring, is usually recommended only at 3-, 6- and 12-months following surgery . OutcomePre-operative DTI profiles offered insights into the differing white matter injury patterns found in NPH. Paradoxically, despite the pre-operative DTI evidence of stretching in the posterior limb of the internal capsule, the gait disturbance of NPH is not accompanied by any signs of a pyramidal disturbance and there are only subtle effects on hand function. Central motor conduction is unaffected in NPH. Despite the widespread DTI changes in the corpus callosum, there is no evidence, except for two case reports, of neuropsychological features of a callosal disconnection syndrome. The cognitive profile of NPH is subcortical in nature; this may be consistent with the subtle white matter injury patterns seen. There are published reports of changes in volume of relevant structures, including the hippocampus and fornix, as well as the caudate nucleus, but the mechanisms of these changes are unclear , 78. AltThe study presents a dataset acquired using modest DTI specifications. This is consistent with other published studies in literature demonstrating similar findings , 80–82 aa priori in patients pre- and post-surgical intervention, that would also allow comparisons of patients against themselves as controls. The results indicate that the DTI methodology using ROIs is able to demonstrate white matter injury in NPH patients compared to healthy controls. ROIs were chosen to minimise the well-known problems posed by crossing fibres and CSF contamination [The degree of ventriculomegaly also differs greatly between patients in NPH, resulting in technical difficulties in generating accurate data from various white matter quantification techniques that rely on computer-generated masks and normalized brain templates, such as Tract-Based Spatial Statistics (TBSS) and Voxel-Based Morphometry (VBM). In addition, the DTI model needs to be sufficiently subtle such that it is possible to observe differences for ROIs selected mination , 84. Sucmination . Our finFew, if any, neurological disorders in elderly patients have demonstrated improvement to the levels of 60–80% with intervention as reported in NPH . Methods


Meditation is becoming increasingly practiced, especially for stress-related medical conditions. Meditation may improve cellular health; however, studies have not separated out effects of meditation from vacation-like effects in a residential randomized controlled trial. We recruited healthy women non-meditators to live at a resort for 6 days and randomized to either meditation retreat or relaxing on-site, with both groups compared with ‘regular meditators' already enrolled in the retreat. Blood drawn at baseline and post intervention was assessed for transcriptome-wide expression patterns and aging-related biomarkers. Highly significant gene expression changes were detected across all groups (the ‘vacation effect') that could accurately predict (96% accuracy) between baseline and post-intervention states and were characterized by improved regulation of stress response, immune function and amyloid beta (Aβ) metabolism. Although a smaller set of genes was affected, regular meditators showed post-intervention differences in a gene network characterized by lower regulation of protein synthesis and viral genome activity. Changes in well-being were assessed post intervention relative to baseline, as well as 1 and 10 months later. All groups showed equivalently large immediate post-intervention improvements in well-being, but novice meditators showed greater maintenance of lower distress over time compared with those in the vacation arm. Regular meditators showed a trend toward increased telomerase activity compared with randomized women, who showed increased plasma Aβ42/Aβ40 ratios and tumor necrosis factor alpha (TNF-α) levels. This highly controlled residential study showed large salutary changes in gene expression networks due to the vacation effect, common to all groups. For those already trained in the practice of meditation, a retreat appears to provide additional benefits to cellular health beyond the vacation effect. More recent systems biology approaches have identified gene regulatory networks associated with a diversity of biological processes, including immune and stress responses, and objectively linked them with disease or salutary states.7Ancient practices such as yoga and meditation have long been thought to combat stress and promote longevity, although empirical evidence for effects on aging processes under highly controlled experimental conditions is lacking. Further, it is inherently difficult to assess effects of meditation apart from simple relaxation. Advances in the understanding of the biological bases of aging enable better assessment of acute effects of salutary interventions on biomarkers of aging. For example, impaired regulatory systems leading to systemic inflammation, and excessive stress responsivity, are related to biological aging6 This bioinformatics approach provides an unbiased view of the immune system profile, and can be linked to changes in environmental conditions.7 These network approaches, while often applied to identifying disease profiles, can be used to identify salutary states as well, such as that which might result from intensive meditation. In addition to high-dimensional molecular data such as gene networks, blood-based biomarkers can provide an integrated overview that indexes biological aging. Telomere length predicts both cellular health and disease in rodent models and humans.8 Shorter telomeres predict onset of cardiometabolic diseases of aging.9 Chronic stress is associated with higher inflammation, shorter telomeres, and lower activity levels of telomerase, the cellular enzyme that elongates telomeric DNA.11 Levels of amyloid beta (Aβ) proteins circulating in the blood appear to be stress-related in rodent models12 and may be affected by stress reduction, and greater Aβ42/Aβ40 ratios are associated with lower risk of dementia.13Integrated systems biology approaches can identify gene regulatory networks, such as immune, stress and other regulatory responses, and link them with physiologic states.16 Preliminary evidence suggests that meditation-based interventions may slow cellular aging rates by increasing telomerase activity, but many such studies lacked an active control group.18 Recent randomized trials in breast cancer suggest that long-term intensive meditation interventions might have positive effects on telomerase activity. One found that mindfulness-based and supportive expressive therapies were associated with telomere maintenance, compared with a ‘treatment as usual' control group.19 A second study found that mindfulness-based stress reduction was associated with increases in telomerase after 3 months.20 Long-term mind–body interventions, including tai chi, yoga and meditation, have been associated with gene expression (GE) changes associated with inflammatory pathways22 as reviewed elsewhere.24Various types of meditation have been shown to improve well-being among different populations such as physicians and the general public.25 Another study comparing experienced meditators to novices after 8  hours of meditation examined GE changes specific to epigenetic regulatory enzymes. Changes were found only in the experienced meditators.26 Another study found threefold changes in GE in the immune cells after yoga versus a control movement program.27 These studies suggest that there are greater changes in experienced meditators than in novices after one bout. However, none of these studies examined people in more controlled residential vacation settings, where larger changes can occur in periods as short as a week. One limitation to meditation studies is that short-term relaxation as a control condition may not lead to the powerful changes that prolonged vacation could. No studies we are aware of have examined how a meditation retreat may affect GE above and beyond a relaxing vacation. Further, none of these previous studies took a systems biology approach by examining covariation across GE patterns (versus specific gene pathways).Short-term interventions have examined changes after one session of meditation or yoga. One study compared GE changes in experienced versus novice meditators after one session. They found changes in both groups in inflammation, energy metabolism, mitochondrial function and telomere maintenance, but experienced meditators had greater changes.28 and greater changes in GE than novices, after meditation,25 we also recruited a third comparison group of experienced meditators. Our design allowed us to study the effects of meditation independent of the vacation effect, as well as to compare the effects of acute intensive meditation in regular meditators versus those newly trained in meditation.Here we examined how exposure to a short-term intensive residential meditation retreat affected biomarkers of aging and more general regulatory networks defining a wide array of biological processes. A residential retreat provides intensive daily exposure in a controlled environment but has the added ‘vacation' effect of taking people away from the demands of their daily lives, which alone might affect regulation of stress pathways. Therefore, it is critical to compare the effects of a meditation retreat with an active randomized control group. Because regular meditators may have differences in brain function and structure, as suggested by meta-analyses,The Retreat and Relaxation Study, a randomized trial, compared the effects of vacation at a resort to training in a meditation/yoga retreat at the same resort (La Costa Resort and Spa). Healthy women aged 30–60 were recruited mostly from the greater San Diego area and San Francisco and randomized to a vacation arm or novice meditator arm. A comparison group of regulator meditators was recruited from women aged 30–60 who had already enrolled in the retreat. The retreat goal was to promote an intensive period of learning and psychological change. The vacation group was hosted at the same resort, but they did not participate in any retreat activities. They reported on well-being immediately after (on day 5), 1 month later, and 10 months later to assess maintenance of benefits.Measures including psychological measures  and detailed methods for the blood draw, processing, biomarker assays and RNA sequencing of blood samples are described in the https://www.synapse.org/#!Synapse:syn7058059.All codes used in the analyses described herein are available upon request. The RNA sequencing data and appropriate descriptions are freely available on the Synapse platform by Sage Bionetworks: 28 and may therefore respond differently to the retreat.To conduct our study, we chose a 1-week retreat offered regularly by the Chopra Center for Wellbeing  at a vacation resort (OMNI La Costa Resort and Spa). Whereas the Chopra Center retreat reflects just one of the many different types of meditation that could be studied, the vacation resort environment at this center allowed us to attempt to distinguish between molecular/biomarker effects induced by a more relaxed, vacation-like environment, versus those induced by the practice of meditation and related activities specific to the meditation arm of the study. We randomized female volunteers who were ‘non-meditators'—having little to no previous experience with meditation—to either the resort only (herein referred to as the vacation arm of the study) or to the retreat (herein referred to as the novice meditation arm) during the same week. The vacation arm serves as a relaxation control for the meditation arms of the study, which take place at a vacation resort, given those in the vacation arm experience all of the amenities of the vacation resort and are at least partially removed from the demands of their daily lives. Although it was not possible to blind participants as to the arm in which they were enrolled, the vacation arm nevertheless affords the possibility of helping tease apart a vacation effect from a meditation effect. The scoring of a comprehensive set of molecular features in this setting provides a more objective assessment of the impact of the different interventions, and the biological processes that were perturbed as a result. In addition to the vacation and novice meditation arms, we recruited a third comparison group of female regular meditators who had already signed up for the retreat for the same week at the resort. This design allowed us to study the effects of meditation independent of the vacation effect, as well as to compare the effects of acute intensive meditation in regular meditators versus those newly trained in meditation. Regular meditators may have differences in brain function and structure, as suggested by meta-analysesThe changes in psychological well-being scores  indicateTo characterize the impact of vacation versus meditation on regulatory networks in blood, we computed three different GE signatures: (1) changes across all treatment groups between the baseline and follow-up time points, and (2 and 3) differences between the regular meditators versus the vacation and novice meditator groups at the follow-up time points. We also compared changes in the vacation versus novice arms at the follow-up time point, and, notably, there was not a significant differential GE signature beyond what would be expected by chance, suggesting that these groups changed in similar ways . This signature was highly conserved among the vacation, novice and regular meditator groups  involved in this pathway were significantly decreased . Other notable genes that were suppressed included stress-responsive genes, such as MME  and FOXO3 (paired t-test=4.89 and P=5.21E−06).As shown in r groups . For thoTo assess the predictive power of the GE changes between the groups, we employed machine-learning procedures to construct classifiers to predict group membership based on pre- versus post-intervention GE (vacation effect) and regular versus non-regular post-intervention GE data (meditation effect). We constructed classifiers to distinguish between the baseline and post-intervention states  and achiTo understand the molecular architecture of the relaxation and meditation GE signatures, we employed an integrative analysis strategy. GE traits from our study were used to construct a weighted coexpression network from which coherent modules of interconnected genes were identified . A broadP=2.1e−60). Translational initiation, protein targeting to the endoplasmic reticulum, translational elongation, viral GE and viral infectious cycle are all closely related processes that are similarly significantly enriched. These pathways were all downregulated in the regular meditator group versus the resort group post-intervention. To identify the hierarchical themes of the most highly enriched pathways, Revigo was used to map out meta-pathways spatially29  were enriched for the meditation signature, one module in particular  stood out as it was >50-fold enriched for pathways related to translation, transport and assembly of proteins and protein complexes, as well as processes relating to viral infection . The GO tially29 .33 constructed from a larger independent blood GE study in which hundreds of individuals were GE-profiled and genotyped . The vacation subnetwork  was downregulated in the post-intervention time point relative to baseline. In the vacation subnetwork MME is linked to NFIL3, a gene found on activated T cells, natural killer cells and mast cells. Another interesting local network structure is centered around CLU, a gene shown to be involved in AD based on genome-wide association studies, with genes in the integrin family, involved in cell adhesion, clustered around CLU.Of the 914 genes in the brown, pink, purple and red modules that map to the Bayesian network, 648 are in the 924-gene vacation subnetwork, a 6.1-fold enrichment over what would be expected by chance (Fisher exact test bnetwork  was enriP<1e−300). For the meditation subnetwork -binding protein that colocalizes with U3 RNA (MIM 180710) in the nucleolus and is required for rRNA maturation and generation of 18 S rRNA.35 TNFRSF10D is also linked to this subnetwork and is involved in apoptosis and cell senescence.Similarly, of the 669 genes in the black, blue, salmon and tan modules that map to the Bayesian network, 508 are in the 836-gene meditation subnetwork, a 7.3-fold enrichment over what would be expected by chance . CLU is a confirmed AD gene from genome-wide association study, and encodes apolipoprotein J, which has been shown to chaperone re-entry of Aβ into the brain following export of the peptide from the brain into the blood.49 Interestingly, genes encoding two peptidases known to degrade Aβ were also decreased in expression in blood pre–post intervention as part of the vacation effect: MME (P=0.00019) and ECE1 (P=0.0037). These data may reflect that there was less need for these peptidases, perhaps owing to lower plasma Aβ40 levels. Finally, expression of the known early-onset familial AD gene, PSEN1, was also decreased as part of the relaxation effect. PSEN1 encodes the γ-secretase needed to produce the Aβ peptide.The increase in the Aβ42/Aβ40 ratio (driven by a decrease in plasma Aβ40 levels) could also be related to the observed decrease in expression of The vacation group showed a statistically significant increase in circulating TNF-α compared with the regular meditators, and a marginally significant increase when compared with novice meditators. Reasons for this increase could include those in the vacation group having an acute stress-related inflammation response to the blood draw, overexposure to sun or some other exposure that can lead to acute physical stress such as exercise. All participants ate a healthy ayurvedic diet, which is thought to be anti-inflammatory.23 Three of these studies found decreased NF-KB and no strong evidence for changes in interleukin inflammatory markers (interleukin-6) or TNF-α, but some evidence for a decrease in CRP and increases in telomerase. Our findings are consistent with this review. We also did not find decreases in circulating inflammatory cytokines but rather slight increases, particularly in the vacation group. We did not find a decrease in NF-KB specifically; however, we did find suppression in large gene networks known to be related to inflammatory processes. We found a trend for increases in telomerase activity in the regular meditator group.One of the most comprehensive reviews on the effects of mindfulness meditation on immune parameters was recently conducted across 20 randomized clinical trials.Our study has several limitations. The changes in biomarkers and GE patterns appear salutary; however, no studies have tracked these markers to see how well they predict disease or longevity in a healthy sample. The sample size is relatively small and powered only to detect medium-to-large effects. Replication of these findings with larger controlled studies will be necessary. In addition, comparing regular meditators to non-meditators is somewhat problematic, given regular meditators likely differ on many lifestyle factors that make such comparisons complicated. For example, diet, exercise regimen and stress-reduction activities such as meditation likely differ between these groups and may not only lead to baseline differences in molecular and higher-order systems, but may prime such systems to respond differentially to resort and meditation interventions. In addition, the meditation arm of this study was not strictly sitting meditation, but also included yoga postures and self-reflection exercises and lectures. Further, whereas our design did attempt to control for the vacation effect, we did not randomize regular meditators to the vacation and meditation arms, making it difficult to interpret the changes observed in the regular meditators, given they were compared with a control group comprising non-meditators. A balanced design that included randomization of regular meditators as well as non-meditators would be useful in future studies to better localize the effects driving the expression changes. Despite these limitations, our study provides a strong distinction between beneficial effects of short-term relaxation typical of a vacation versus acute intensive meditation.In summary, our results point to both a significant ‘vacation effect' that benefitted all groups, and a suppression of stress-related responses and immune function related to acute-phase wound healing and inflammation. We also identified a ‘meditation effect' within the regular meditator group, characterized by a distinct network of genes with cellular functions that may be relevant to healthy aging, and this network was associated with increased expression of a number of telomere maintenance pathway genes and an increase in measured telomerase enzymatic activity. This study provides a strong distinction between beneficial effects of short-term relaxation typical of a vacation versus acute intensive meditation for regular meditators. Future studies expanding upon these results will be critical for further understanding lifestyle acute adaptations capable of promoting stress reduction and overall health and well-being.


Studies on the effects of moderate prenatal exposure to cadmium (Cd) on birth outcomes have been contradictory and it has been suggested that effects may be partly masked by sex‐specific effects. Our aim was to examine the association of Cd exposure in a large group of pregnant women with birth outcomes in the whole group of participants and by sex.n = 4191). Data collected on the infants included anthropometric variables and gestational age at delivery. Data were analysed using SPSS v18.Pregnant women were enrolled in the Avon Longitudinal Study of Parents and Children (ALSPAC). Whole blood samples for singleton pregnancies with a live birth were analysed for Cd  and crown–heel length  in adjusted regression models. On stratification by sex, maternal blood Cd level was adversely associated with birthweight , head circumference , and crown–heel length  in girls but not in boys in adjusted regression models.In these pregnant women with moderate prenatal Cd exposure there evidence of adverse associations with birth anthropometry variables in the whole group. However, there was evidence of associations with anthropometric variables in girls that were not evident in boys. Sex‐specific effects require further investigation in large cohorts as a possible contributor to the lack of associations generally found in mixed‐sex studies. Cadmium (Cd) is a non‐essential cumulative toxic metal that is widespread in the environment. Occupational exposure is generally associated with battery manufacture and recycling, and non‐occupational exposure arises through fossil fuel combustion, waste incineration, and manufacturing processes including those for cement, iron, and steel. Smoking tobacco, however, is the most important source of Cd in the general population: cigarettes contain about 0.5–1.5 μg Cd/cigarette, with 20–50% of this transferred directly to the circulation.Adverse health effects from Cd exposure include kidney, breast, prostate, and lung cancer; cardiovascular disease; and osteoporosis. These diseases may be related to the role of Cd as an endocrine disruptor through interaction with hormone signalling pathways.The aims of this study were: (1) to evaluate the effect of moderate prenatal exposure to Cd on a range of birth outcomes  independently of active and passive smoking status in a large cohort of pregnant women, (2) to evaluate sex‐specific effects of maternal Cd exposure on these birth outcomes.www.bris.ac.uk/alspac and the study website contains details of all the data that are available through a fully searchable data dictionary . Ethics approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees.The sample was derived from the ALSPAC study, a population‐based study investigating environmental and genetic influences on the health, behaviour, and development of children. This database provided an opportunity to include a large number of participants and includes a wide range of social and demographic information to enable the most appropriate selection of covariates. All pregnant women in the former Avon Health Authority with an expected delivery date between April 1991 and December 1992 were eligible for the study; 14 541 pregnant women were enrolled, resulting in a cohort of 14 062 live births. The social and demographic characteristics of this cohort were similar to those found in UK national census surveys. Further details of ALSPAC are available at Whole blood samples were collected in acid‐washed vacutainers  by midwives as early as possible in pregnancy (median of 11 weeks’ gestation (IQR 9–13 weeks) with a mode of 10 weeks). Whole blood samples were stored in the original tube at 4°C at the collection site before being transferred to the central Bristol laboratory within 1–4 days. Samples were at ambient temperature during transfer (up to 3 h). They were then stored at 4°C until analysis. Details of the analysis have been reported.Newborn head circumference and crown–heel length were measured by trained study staff where the mother gave permission, or if these data were missing, the values were extracted from the medical records by trained study staff. Birthweight was derived from obstetric data and from central birth notification data: where values disagreed by <100 g then the lowest value was accepted; if the values disagreed by >100 g then the value was coded as missing. Study staff were blinded to the maternal blood Cd. Length of gestation was based on last menstrual period date, ultrasound assessment or other clinical indicators. Where there was conflict of ≥14 days between the maternal report and ultrasound assessment, an experienced obstetrician reviewed the clinical records and made a best estimate. Low birthweight (LBW) was defined as birthweight <2500 g and preterm birth as <37 weeks gestation.http://www.bristol.ac.uk/alspac/researchers/questionnaires/).The mothers received four postal self‐completion questionnaires during pregnancy. The questionnaires are available from the study website  and on whether the partner smoked (yes/no). If gestation was >14 weeks at enrolment, the questionnaire was completed 4–7 weeks after enrolment.Participants were excluded from the study if they did not have a singleton live birth . Statistical analysis was done with SPSS version 23 . Values are reported as mean±SD. Chi‐square tests were used to analyse differences in categorical data, and ANOVA was used to compare continuous values by tertiles of B‐Cd.The main analyses were conducted for term deliveries only (≥37 weeks). Additional analyses were conducted for all gestational ages (results shown in supporting information Tables). Univariate and multivariable linear regression models were used to examine the relationship of B‐Cd with birthweight, head circumference, and crown–heel length. Logistic regression analysis was used to examine the effect of the binary outcome variable LBW (<2500 g) by tertiles of maternal B‐Cd . Model 1 included the following confounders: maternal age, maternal height, maternal pre‐pregnancy body mass index, maternal educational attainment, parity (0 versus ≥1), alcohol consumption (number of units per week), and sex of the baby. These factors were chosen as being consistently associated with birthweight, length of gestation, and fetal growth retardation as well as other measures including low birthweight, preterm delivery, and small‐for‐gestational‐age (SGA) at birth as well as B‐Cd. We did not include maternal ethnicity in any of the models because of the low numbers of ethnic minority participants included in the sample (non‐white 2.4%). Model 2 also included maternal smoking (yes/no); partner smoking (yes/no) was added for model 3. Gestational age was not included as a confounder in the association between prenatal exposure and birth outcome as it is considered to be a collider in the association between environmental exposure and birth outcomes, and could therefore mask associations.We undertook three further analyses. (1) Since there was collinearity between smoking and B‐Cd, models 1–3 for all births were repeated after stratification for smokers and non‐smokers. (2) The mediating effect of Cd in the association between smoking and anthropometric outcomes was assessed with models of the associations with and without adjustment for the maternal B‐Cd level. (3) Sex‐specific effects of maternal B‐Cd were evaluated by repeating models 1–3 for all births after stratification by the sex of the offspring.Regression diagnostics were used to check that the models fitted the observed data well and to identify any cases that had undue influence on the model.There were 4191 singleton livebirths included in the study, of which 3838 were a term birth and were included in the main analyses. Mothers with data on B‐Cd were broadly similar to those without, except that they had a higher educational attainment and were slight older.P < 0.001).The mean maternal B‐Cd level was 0.56 ± 0.62 (range 0.14–6.30)  μg/L. As reported previously, the mean B‐Cd level was similar to those reported in other developed countries.P ≤ 0.001; Table In univariate analyses with tertiles of B‐Cd, there was strong evidence for negative associations of maternal B‐Cd with birthweight, head circumference, and crown–heel length .There were strong associations between maternal smoking and anthropometric outcomes  in adjusted linear regression models Table  for the On stratification by sex, birthweight, head circumference, and crown–heel length were significantly adversely associated with maternal B‐Cd in girls but not in boys in adjusted models Table . The intThere was evidence of associations between moderate maternal B‐Cd level and some birth outcomes  but not with others  in adjusted regression models in a large cohort of women. However, on stratification by sex there were significant adverse associations between maternal B‐Cd and birthweight, head circumference, and crown–heel length in girls but not in boys. Although maternal B‐Cd was associated linearly with an increase in the number of cigarettes smoked per day, with an additional effect of partner smoking, in univariate analysis, there was no evidence that B‐Cd levels in either smokers or in non‐smokers were associated with adverse birth outcomes. There was no evidence that maternal B‐Cd mediated adverse associations between smoking and birth outcomes.11β‐HSD2 and 3β‐HSD, which control the transfer of glucocorticoids to the fetusThe mechanisms for the putative effects of Cd on birth outcomes may be related directly to toxic effects on the fetus and/or mediated through toxic effects within the placenta. Although the placenta may act as a partial barrier to Cd being transferred to the fetus, it is likely that at least some Cd accumulates in the placenta.et al.Despite these theoretical effects, the evidence from epidemiological studies for negative associations of maternal Cd status with birth outcomes, particularly for outcomes other than birthweight, is mixed. With regard to birthweight, the results of studies of associations with maternal or cord blood Cd and have been conflicting. In the largest study to date, Wang et al.To our knowledge, only one previous study has stratified the data by smoking status: Menai et al.et al.11β‐HSD2 activity, with a consequent increase in cortisol level; (2) differential Cd interference with the IGF axis, as there is some evidence that levels of IGF subtypes are sex‐specific; (3) the male skeleton may be more resistant to Cd‐induced bone damage through differential effects on bone mineralization, bone density, and bone mineral content; (4) there may be effects on in utero sex‐specific programming that contribute to the vulnerability of the fetus to environmental stressors. As suggested by Rollin et al.It is possible that associations of birth outcomes with maternal B‐Cd in mixed‐sex groups could be masked by opposing effects in female and male offspring. It has been suggested that associations, particularly with anthropometric variables, tend to be adverse in girls but beneficial in boys, as was suggested in the present study for head circumference and crown–heel length. This was first proposed by Kippler This study has a number of strengths. (1) The study involved a large number of pregnant women with both prenatal B‐Cd and birth outcomes. (2) The prenatal exposure was B‐Cd measured in the first half of pregnancy, in contrast to studies that have relied on cord blood Cd or Cd in other matrices such as hair or urine. (3) The data on maternal smoking were well characterized. (4) We were able to include data on passive smoking. (5) We were able to differentiate effects in female offspring from male offspring. There are some limitations to the study. (1) Although we were able to account for many possible confounders in our analyses, there are likely to be others that were unable to adjust for. These include the effects of other environmental pollutants, such as polychlorinated biphenols.In accordance with putative mechanisms of the toxic effects of Cd on birth outcomes, we found evidence for independent associations of maternal B‐Cd level with birthweight and crown–heel length in adjusted linear regression models, but not with head circumference. There was no evidence of an increased odds of having a low birthweight baby in adjusted logistic regression models. When the models were repeated after stratification into smokers and non‐smokers, there was evidence for an adverse association with birthweight in smokers. Maternal B‐Cd was not a strong mediator in the associations between smoking and any of the pregnancy outcomes. However, there was strong evidence of differential effects in female and male offspring on anthropometric variables . We conclude that sex‐specific effects require further investigation as a possible contributor to the lack of associations found in most mixed‐sex studies.Figure S1. Flow chart for study.Table S1. Summary of studies on maternal Cd status and birth outcomes.Table S2. Characteristics of pregnant women in ALSPAC included in the present study .Table S3. Effect of active and passive smoking on maternal B‐Cd levels (μg/L) in ALSPAC.Table S4. Birth outcomes by maternal B‐Cd tertile in ALSPAC.Table S5. Associations of maternal B‐Cd with birth outcomes in ALSPAC (linear regressions).Table S6. Associations of tertiles of maternal B‐Cd with birth outcomes in ALSPAC (logistic regressions).Table S7. Linear regression of maternal B‐Cd with birth outcomes stratified by smoking in ALSPAC.Table S8. Logistic regression for tertiles of maternal B‐Cd with birth outcomes stratified by smoking in ALSPAC.Table S9. Mediation of maternal B‐Cd in associations between smoking and birth outcomes in ALSPAC.Table S10. Sex‐specific differences in birth outcomes in association with maternal B‐Cd levels in ALSPAC.Click here for additional data file.