Early childhood factors can have persisting effects on development and cognition in children. We propose to explore the trends of Fe deficiency and Pb toxicity in early childhood and their association with child development at 2 years of age and cognition at 5 years.Longitudinal birth cohort study.Urban slum, Vellore, India.Children enrolled at birth were followed up regularly in the first 2 years with developmental and cognitive assessments at 2 and 5 years of age, respectively.The birth cohort enrolled 251 children with 228 children followed up at 2 years and 212 at 5 years of age. Fe deficiency (ID) was highest at 15 months of age and improved subsequently at 24 months. Blood Pb levels (BLL) remained high at all age groups with an increasing trend with age; 97 % at 36 months having high BLL. Persistent high mean BLL at 15 and 24 months had negative association with both cognition and expressive language raw scores of 24 months, while high mean BLL at 15, 24 and 36 months had no significant association with any of the domains of cognition at 5 years of age. Early childhood cumulative body Fe status at 7, 15 and 24 months did not show any association with child development at 2 years, but was associated with verbal, performance and processing speed components of cognition at 5 years.Optimising body Fe status and limiting Pb exposure in early childhood can augment child development and school entry cognition. There are many genetic, environmental and epigenetic factors that affect child’s development, and it is challenging to identify a single factor, the deficiency of which is common, easy to detect and treat, and amenable for community-level interventions.It is now recognised that the ‘first 1000 days of life’, from conception to the second birthday, are critical for brain development. This has resulted in an extensive exploration of early childhood factors to understand their potential impact on child development and sequentially human potential,3. Fe deficiency (ID) is considered as the most common nutritional deficiency among all age groups and is prevalent across all social strata,4. Fe is vital, not just for the synthesis of Hb but also for the non-haematological effects on mitochondrial and neuroendocrine function. ID therefore affects cognition and behaviour in children, and these changes can be long term. Although there are studies evaluating anaemia and Fe status and their effects on cognition, there is a dearth of evidence, of the long-term effect of ID in early childhood, especially from low–middle income countries.Micronutrient deficiencies such as that of Fe and I, especially in early infancy, can have long-term effects on cognition and behaviour, probably due to their impact on the rapidly developing brain during the ‘first 1000 days of life’. With increasing recognition of the adverse effects of high Pb levels on young brains, there has been a revision of the cut-off for elevated blood Pb levels (BLL) from 0·48 µmol/l (10 µg/dl) to 0·24 µmol/l (5 µg/dl) by the American Academy of Pediatrics,7. Background deficiency of micronutrients particularly that of Fe can modify the impact of exposure to high levels of Pb through pica and competitive inhibition of intestinal absorption,9. A previous study from the same birth cohort had reported high BLL at 15 and 24 months of age in children. The objective of the current study was to evaluate the trends of both Fe and Pb status in early childhood and the association of cumulative early childhood Fe and Pb status on development at 2 years of age and cognition at 5 years of age.In addition to micronutrient deficiency, Pb exposure in early childhood can adversely affect child development including cognition. Even low levels of Pb exposure during the early childhood brain development stage can have lasting effects on cognition and behaviour. The study site in India was eight adjacent urban slum dwelling areas, located in Old Town, Salavanpet and neighbouring areas in Vellore, South India, with a population density of 42 000/km2, compared with 6200/km2 in Rio de Janeiro and 32 000/km2 in Harlem, NYC. A population of 12 000 was covered for the current study. All pregnant women in the study site were identified by door-to-door survey and invited to participate in the study immediately after the delivery, by consecutive sampling. The enumeration process carried out prior to recruitment provided the probable number of births in the study area based on recent births and the number of pregnant mothers. Using this information, it was planned to recruit more than 200 children over the 2-year period, the target number for each site as per the MAL-ED study protocol . Mothers were screened based on the following exclusion criteria – having pre-existing plans for migration during the study period, having another child from the same family being enrolled in the MAL-ED study, multiple pregnancies, having a neonate with medical complications and unavailability of mothers to provide informed consent. Babies of mothers who consented were enrolled, and their demographic characteristics along with anthropometry measurements were recorded. Babies were enrolled at a mean (range) age of 10·08 (0–16) d. Trained fieldworkers visited their home twice a week for active disease surveillance for the first 2 years of life. The initial birth cohort recruitment was conducted between March 2010 and February 2012 and recruited 251 children, and baseline demographic data of the birth cohort were comparable to that of the study area. The cohort was further followed up at 36 and 60 months of age.The study population was a birth cohort followed up for the Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) Network, a multinational, longitudinal prospective cohort study conducted in eight countries across the world. Cognition or intelligence is defined as a combination of multiple abilities in a child. The original study and further follow-up studies were approved by the institutional review board. The current study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects namely growth monitoring, cognition assessments and blood collection were approved by the institutional review board, Christian Medical College, Vellore, India. Written informed consent was obtained from the parents/caregivers of all the subjects.Blood samples were collected at 7, 15, 24, 36 and 60 months. Samples were tested for Hb ; Fe and ferritin assays  and for BLL . All children underwent a developmental assessment at 7, 15, 24 and 36 months of age using Bayley Scales of Infant and Toddler Development-3rd edition (BSID-III) and cognitive assessment at 5 years of age, using Wechsler Preschool Primary Scales of Intelligence – third edition (WPPSI-III). Child development is defined as the ability of the child in domains of motor, language, cognition, play, social and adaptive skills® Hb 301 kit (azide-methemoglobin method), at the study clinic immediately after blood collection using a drop of blood. One millilitre of the collected venous blood was used for BLL analysis and preserved with K2 EDTA, and the remaining blood was stored in the biochemistry tube for analysis of serum ferritin and transferrin levels. The samples were refrigerated and transported using cold packs to the research laboratory within 2 h of blood collection for further analysis. Serum ferritin, transferrin receptors and Pb levels were tested in the Biochemistry department, Christian Medical College, Vellore. Chemiluminescence immunoassay was done for serum ferritin analysis using Siemens Advia Centaur (Model Siemens Advia Centaur Xp Chemiluminescence immune assay) with 0·5 μg (11·24 pmol) as limit of detection. For estimating transferrin levels, Immunoturbidimetry was performed using Roche Coba C system (model Roche Cobas C 702 Immunoturbidimetry assay) with 10 μg (0·13 µmol) as limit of detection. Using the Graphite Furnace Atomic Absorption Spectroscopy method in M-series Atomic Absorption Spectro-photometer (model Thermo Scientific X series 2.ICP-MS), BLL were estimated with 0·5 μg (0·024 µmol) as limit of detection.Venous blood was collected from children to a maximum quantity of 5 ml at each visit. Hb was analysed using a point-of-care screening tool, Hemocue. The measure was culturally adapted, translated and piloted prior to administration. A single trained psychologist conducted the testing in a standardised child friendly setting in the community clinic for each age group. Around 5–6 % of the tests were video-recorded and centrally reviewed for quality control purposes. For the purpose of current analysis, the 24-month assessment was used, as 2 years denote the completion of infancy and the ‘1000-day window’ period. Receptive and expressive language and cognition domain raw scores were derived for each child from the 24-month assessment. Receptive and expressive language skills measure receptive and expressive abilities of children in primary language and communication areas, while cognition measures sensorimotor development, exploration and manipulation, object relatedness, concept formation and memory.The BSID-III assesses development in the domains of gross motor, fine motor, expressive language, receptive language and cognition between 1 and 42 months of ageThe WPPSI-III can be administered to children aged between 2 years 6 months and 7 years 3 months and assesses cognition in the domains of verbal, performance and processing speed. This measure was adapted centrally for cultural appropriateness and was translated and piloted in local settings before being administered by a single trained psychologist. Verbal, performance and processing speed raw scores were obtained after administration for each child. Ten percentage of these assessments were recorded and reviewed centrally for quality control.. This time of assessment was chosen in the protocol for mothers’ convenience as they would be able to leave their babies with another caregiver around this time, to complete cognitive assessment in the study clinic. To assess the socio-economic status (SES), a socio-economic index was created using Water and sanitation, Assets, Maternal education and household Income (WAMI) and SES of the families was assessed at recruitment and six-monthly intervals as per the study protocol. The WAMI index was validated against prevalence of stunting in all the eight countries involved in the MAL-ED cohort study. The broader aim of the main study was to assess the predictors of early child development, amongst which SES influences child development through both biological and physical home environment,18.Maternal intelligence was assessed at 6–8 months of child age by Raven’s progressive matrices:As serum ferritin levels can be influenced by inflammation, total body Fe levels were calculated from ferritin (F) and transferrin (R) levels as belowPositive values indicated Fe surplus in stores, while negative values indicated deficiency of Fe in tissues.The Data Coordinating Center developed a double data entry database application. Forms were checked for completeness and accuracy of the data by data supervisors before being entered into the application. Discrepancies between the first and second entry were cleared by data supervisors after rechecking forms, and this process of cross validation was done for quality control of the study. The data was analysed using STATA 13 (StataCorp). Missing data values were limited as this was a prospective study with rigorous quality checks. If a child did not have information at one time point, that child was excluded from corresponding analysis and no missing value corrections were applied.sd were used to summarise Hb, Pb, ferritin and transferrin levels measured at each time point. A birth weight <2·5 kg was considered as low birth weight category. We reported children who were anaemic (Hb < 11 g/dl), Fe deficient (blood Fe < 0 mg/kg) and with elevated BLL (>0·24 μmol/l) using proportions. Characteristics of the study children such as sex and SES at 2nd and 5th year of follow-up were compared with baseline data using ‘Chi-square for trends’. Mean values of Hb across the time points  were compared using repeated-measures ANOVA. Bonferroni test was used for post hoc analysis, to compare the Hb levels between any of the two time points measured. Estimates of median serum ferritin, transferrin and BLL across each time point were compared using Friedman’s test, and post hoc analysis between any of the two points was done using Wilcoxon Signed-rank test. Proportions of children with anaemia, ID and elevated Pb levels across each time point were compared using ‘Chi-square for trends’. The proportion with ID in anaemic children was evaluated to understand causes of anaemia in each age group setting. The outcome variables namely child development (BSID-III) measured at 2 years were expressed as raw scores of cognition, expressive language and receptive language domains, and cognition (WPPSI-III) measured at 5 years was expressed as raw scores of verbal, performance and processing speed domains. Median (IQR – interquartile range) was used to summarise the scores under each domain of the outcome variables. Cronbach’s alpha was used to assess the internal consistency within items under each domain of outcome measures to evaluate if the responses provided by the subject remained consistent. Since Fe and Pb levels were analysed at different time points, a summary statistic approach was used and the repeatedly measured information was condensed to a single number per subject,22. Mean body Fe was derived by averaging the body Fe levels at 7, 15 and 24 months, and this measure was considered for analysis at both 2 and 5 years. Mean BLL was derived by averaging the BLL at 15 and 24 months for analysis at 2 years, and those at 15, 24 and 36 months for analysis at 5 years.Mean and r value) of <0·5 in the multicollinearity analysis were retained in the model. The standardised beta coefficient along with 95 % CI of the predictor variables was noted. In the post-estimation analysis, homoscedasticity of the residuals was assessed using Breusch–Pagan test. The level of significance for the test of association was considered at P < 0·05.Linear regression modelling was done to find the predictors of the outcome variables – BSID-III and WPPSI-III scores using separate models. The predictors that were included in the model were – sex of the child, maternal intelligence raw scores, SES, mean body Fe levels and mean BLL. In the bivariate analysis, unadjusted beta coefficient along with 95 % CI was reported for each predictor. A multivariable linear regression was performed using the same set of predictors listed above. Following assumptions of linear regression were tested – normality of the outcome variable using Shapiro–Wilk test and multicollinearity of predictors using correlation matrix were tested. Only those independent variables with the correlation coefficient (n 5), another child of the same family being part of the MAL-ED study (n 8), multiple pregnancy (n 1), presence of pre-existing morbidities in children (n 7) and mothers not available to give informed consent (n 9) and a combination of two or more of the above reasons (n 10). On follow-up, 228 children participated at 2 years of age and 212 children at 5 years of age . Reasons for screen failures included pre-existing plans for migration during the study period (. The mean BMI of mothers was 22 (sd 3·9) kg/m2 with 20·3 and 21·1 % of mothers being underweight and overweight/obese, respectively. The cohort participants were predominantly girls (56 %), and the mean birth weight was 2·8 (sd 0·44) kg. Seventeen percentage of babies were low birth weight and weighed <2·5 kg at birth. The mean exclusive breast-feeding duration was 76·9 (sd 43·8) d.The birth cohort had a mean (range) family size of 5·7 (3–13) and the mean (range) educational year of the head of the family was 6·8 (0–18)P = 1·000) . Although ferritin levels significantly dropped between 7 and 24 months (P < 0·0001), there was no change at 36 months . There was a significant increase in mean BLL across the three time points   Table . SubsequMore than half of the children were anaemic at 7 and 15 months of age, with subsequent improvements in Hb levels at 24, 36 and 60 months. Almost half of the study group (46·1 %) had low Fe status at 15 months of age , 29 (IQR 15–36) and 59 IQR 47–69), respectively, at 2 years of age, while the median raw scores at 5 years of age for verbal, performance and processing speed domains were 39 (IQR 13–69), 48 (IQR 22–75) and 35 (IQR 1–86), respectively  and had an adjusted R2 value ranging between 5 and 7 % . Considering the collinearity between the body Fe and Hb , the Hb was excluded from the final model. High mean BLL of 15 and 24 months was associated with lower cognition and expressive language raw scores at 2 years of age, which remained significant in the multivariate analysis with sex, mean body Fe , maternal intelligence and SES (Table The multivariate models built for each domain of the child development measured at 2 years of age Table  were staP < 0·0001) and had an adjusted R2 value ranging between 10 and 13 % . Body Fe status of early childhood  had a significant association with verbal, performance and processing speed at 5 years of age, which remained significant in the multivariate analysis  conducted during 2015–2016 (NFHS-4) and 2005–2006 (NFHS-3), respectively. Although the prevalence of anaemia had lowered to 58·5 % in NFHS-4, from its previous estimate of 69·4 % in NFHS-3, anaemia continues to be a public health problem in India. Other studies from India also had reported that anaemia was common in the older age group with a prevalence ranging between 27 and 63 %,29. Other causes of anaemia in children from low–middle income countries are hookworm infestation with resultant blood loss, vitamin B12 deficiency manifesting as megaloblastic anaemia, haemoglobinopathy disorders and environmental enteropathy,30,31. However, the association between above-mentioned causes of anaemia and cognitive development was not investigated in the current analysis.This prospective birth cohort follow-up study from urban Vellore provided the trends of Hb, Fe and Pb levels during the early childhood. Further, a negative association of high BLL with cognition and expressive language domains of child development was demonstrated. The current study also showed a negative association of ID with verbal, performance and processing domains of cognition. Retention in the current study was good at all planned time points. There was minimal drop out and 212 children presented at 5 years of age  as the cut-off for high level; but the revised cut-off of 5 µg/dl (0·24 µmol/l) has been used for the current analysis,7,41. This current community-based prospective study shows increasing Pb levels from 15 to 36 months of age, with high Pb levels present in 97 % of children (>5 µg/dl (0·24 µmol/l)) at 36 months of age. This has been reported also from Nepal, in a hospital-based study of 6–36 months old children, where 64·4 % had high BLL of >5 µg/dl (0·24 µmol/l). Risk factors associated with high BLL include low SES, industrial area exposure, playing with dirt and mud, exposure to enamel paints, piped water supply and mud/clay floors of the house; the last two factors being shown as predictors for high BLL in this birth cohort in an evaluation published earlier,38–44. There is evidence suggesting transplacental transfer of Pb, with sixteen (84·2 %) cord blood samples obtained from nineteen mothers, who had high BLL during their antenatal period, had elevated Pb levels. This highlights the possibility of Pb exposure even as early as the in utero phase of life. In the current study, the continued environmental exposure to Pb during early childhood could have caused increasing BLL trends.High BLL as in the current study have been reported from India,9. Heavy metals including Pb share intestinal Fe absorption pathways, where there can be competitive inhibition between Pb and Fe. The classic clinical manifestation of Pb poisoning in children is anaemia, more specifically hypochromic microcytic anaemia. The prevalence and severity of anaemia correlate directly with BLL. Younger and Fe-deficient children are at higher risk of developing Pb-induced anaemia. Pb toxicity results in anaemia through two mechanisms – by causing direct damage to the cell membrane of erythrocytes resulting in haemolysis and through inhibition of the enzyme delta-aminolevulinic acid dehydratase in the Fe-dependent haem synthesis pathway,46. Inhibition of delta-aminolevulinic acid dehydratase enzyme not only results in anaemia by affecting haem synthesis but also results in the accumulation of haem precursors such as aminolevulinic acid, which can cause harmful effects to the nervous system,47.There can be interactions between Pb and Fe. ID can cause pica, which can exaggerate Pb exposure through mouthing of inanimate objects,49. Pb even in smaller dose exposure is toxic to the nervous system and can impair cognition and behaviour; thus the recommendation that there is no safe BLL,41. Pb interferes with ion channels in the brain by its function as a Ca analogue,46. In addition, Pb increases oxidative stress by generation of reactive oxygen species and inactivates antioxidants such as glutathione by binding to them,51. Pb can accumulate over time as shown in the current study and can have a negative association with cognition and learning such as impaired visual-motor integration, longer reaction time and reading disability in school age years; which persist into young adulthood,49.In the current study, high mean BLL over 15 and 24 months had negative association with both cognition and expressive language raw scores of 24 months. Literature shows that early childhood Pb exposure can adversely affect later age cognitive capability – a doubling of BLL or tooth Pb levels at 2 years of age can cause a mean deficit of 1–2 intelligence quotient points at 5 years of age,52. Early onset ID through its impact on the activity of Fe containing enzymes can affect neuronal myelination, mitochondrial function and neurotransmitter metabolism of developing brain, which can have long-term effects on cognition and behaviour of children,4,34,53. Subsequent Fe supplementation beyond the early childhood can optimise body Fe stores, but cannot neutralise the impact of early onset ID on neurodevelopment, cognition and behaviour,53,54. ID in school age years can also affect cognition, as per other Indian studies,28,30,55. Among cognitive functions, ID impairs executive function the most, which includes but not limited to attention, inhibitory control, planning ability and working memory,4,36,54. ID can also cause concentration and attention problems, tiredness and low mood in school-age years; which can improve on appropriate Fe supplementation,54. ID without anaemia in women of reproductive age group was shown to affect their executive function, indicating the need to evaluate ID on its own without the ramification of presence or absence of anaemia. Antenatal or early childhood Fe supplementation shows inconsistent effects on both early- and long-term cognition in children,57,58.The finding of the current study is that early childhood cumulative body Fe status had no association with development at 2 years of age, but was associated with verbal, performance and processing speed components of cognition at 5 years. This may be due to the fact that cognition is a stable construct at 5 years as frontal lobes are better myelinated and mature at this age but also by providing optimal macro- and micro-environments including appropriate play materials and stimulating experiences,60,61. Improvement in SES factors such as access to safe water and sanitation, better maternal education status and improvement of family economy can offer better experiences for children to grow and develop. Repeated diarrhoeal illnesses predisposed by lack of safe water and sanitation facilities in the early childhood can have direct negative impact on child development and cognition. Indirect impact of diarrhoeal illnesses on cognition is through manifestation of nutritional deficiencies following these illnesses, which in turn affects cognitive development. In addition to these infections, exposure of toxins such as Pb can further affect the cognition,61. Girls tend to have better speech and expressive language skills than boys at 2 years of age, as shown in the current analysis, probably due to better environmental interactions,63.Other factors that have association with development and cognition in the current study are maternal cognition, SES factors and sex. Maternal intelligence can affect child cognition not just by genetic influenceThe current study has strengths such as longitudinal birth cohort follow-up, standardised developmental and cognitive assessments by a single trained psychologist, rigorous quality control with internal and external reviews, standardised laboratory assessments and adjustment for SES. However, regression models could explain only up to 13 % of the variance associated with the predicted outcomes, highlighting the presence of other factors that could have influenced the outcome. A comparatively smaller sample size prevented a detailed analysis of other factors within the model. Inclusion of factors such as years of preschool education could have made the model more explanatory. Due to the collinearity between serum Fe and Hb levels, the latter was not included in the regression; which can also be a limitation to the current study. Despite these limitations, this prospective birth cohort study showed that high BLL in early childhood was negatively associated with development at 2 years of age and the association of early childhood ID with cognition at the age of 5 years. Future studies can explore the contribution of other early childhood micronutrient deficiency and environmental toxicity to development and cognition, and the ideal timing of nutritional interventions such as Fe supplementation and fortification.This longitudinal birth cohort shows maximum ID at 15 months of age and increasing BLL with age in a low–middle income country setting.Early childhood ID shows a negative association with verbal, performance and processing speed domains of cognition at 5 years of age, while high BLL was negatively associated with cognition and expressive language at 2 years of age.


Enhanced recovery after surgery (ERAS) protocols are evidence-based, multimodal and patient-centred approach to optimize patient care and experience during their perioperative pathway. It has been shown to be effective in reducing length of hospital stay and improving clinical outcomes. However, evidence on its effective in liver surgery remains weak. The aim of this review is to investigate clinical benefits, cost-effectiveness and compliance to ERAS protocols in liver surgery.A systematic literature search was conducted using CINAHL Plus, EMBASE, MEDLINE, PubMed and Cochrane for randomized control trials (RCTs) and cohort studies published between 2008 and 2019, comparing effect of ERAS protocols and standard care on hospital cost, LOS, complications, readmission, mortality and compliance.p < 0.00001) compared to the standard care group. Fewer patients in ERAS group experienced complications . Hospital cost was significantly lower in the ERAS group .The search resulted in 6 RCTs and 21 cohort studies of 3739 patients (1777 in ERAS and 1962 in standard care group). LOS was reduced by 2.22 days in ERAS group (MD = −2.22; CI, −2.77 to −1.68; Our review concluded that the introduction of ERAS protocols is safe and feasible in hepatectomies, without increasing mortality and readmission rates, whilst reducing LOS and risk of complications, and with a significant hospital cost savings. Laparoscopic approach may be necessary to reduce complication rates in liver surgery. However, further studies are needed to investigate overall compliance to ERAS protocols and its impact on clinical outcomes. ERAS protocols have been implemented in various surgical specialties especially colorectal surgery from the early 2000s. However, due to patient safety concerns and high complication rates, the first cases of ERAS protocols in liver surgery only appeared in the scientific journals in 2008.3The concept of developing a ‘multimodal approach’ to accelerate recovery and rehabilitation after surgery was first developed by Kehlet.4 In 2016, the ERAS Society published its first guideline for perioperative care in liver surgery to add to existing ERAS guidelines regarding other surgical specialities.5 This particular guideline consists of 23 items including preoperative counselling, preoperative carbohydrate loading, perioperative nutrition, avoidance of bowel preparation, no routine use of surgical drain, thromboembolism prophylaxis, antibiotic prophylaxis, minimally invasive approach, intraoperative fluid restriction, multimodal analgesia, prevention of hypothermia, early oral fluid and normal diet intake, glycaemic control, prevention of delayed gastric emptying, stimulation of bowel movement, early mobilization, prevention of postoperative nausea and vomiting, fluid management and systematic audit. Majority of the recommendations were based on evidence from colorectal surgery due to limited evidence in liver surgery. With entirely different patient cohorts between colorectal and liver surgery, their different morbidities and therefore physiological stress on patients, it is questionable whether ERAS principles used in colorectal surgery can be truly extrapolated to liver surgery.The ERAS programme is an evidence-based, multimodal and patient-centred approach to optimize patient care and experience during perioperative care.6 With recent advancement in surgical techniques and improvement in perioperative care management, a mortality rate of less than 5% is now achievable.7 Moreover, several reviews concluded that the implementation of ERAS protocols is associated with a significant reduction in length of hospital stay and postoperative complications without increasing mortality and readmission rates.14 However, evidence on cost-effectiveness and compliance to ERAS protocols in liver surgery is limited. Two previous meta-analyses have demonstrated a significant reduction in hospital expenditure following implementation of ERAS protocols in liver surgery.10 The conclusion reached by these reviews was either based on low-quality RCTs and cohort studies without conducting a separate meta-analysis of RCTs and cohort studies. Furthermore, to date, no review has investigated overall protocol compliance which is a key factor to delivering a successful implementation of ERAS protocols. The aim of this review is to investigate clinical benefits, cost-effectiveness and compliance to ERAS protocols in liver surgery.Traditionally, liver surgery is known to be associated with high complication and mortality rates.15 A systematic search was conducted in March 2019 on the following database: CINAHL Plus, EMBASE, MEDLINE, PubMed and Cochrane for studies published between 2008 and 2019. The search was limited to English language. Reference lists of relevant RCTs and systematic reviews were searched for eligible studies. The search terms such as ‘enhanced recovery’, ‘fast track’, ‘ERAS’, ‘perioperative care’, ‘enhanced rehabilitation’, ‘liver resection’, ‘liver surgery’, ‘hepatic resection’, ‘hepatobiliary’, ‘HPB surgery’, ‘hepato-pancreato-biliary’ and ‘hepatectomy’ were applied using Boolean operator (OR and AND).This review was conducted in accordance with PRISMA  guidelines for meta-analysis.RCTs and non-RCTs studies were eligible for inclusion if they all meet the following criteria, (1) studies of adult patients undergoing liver surgery, (2) compared ERAS to standard care and (3) report on at least one of the following outcomes: length of hospital stay (LOS), complication rate, hospital cost, readmission rates and mortality rates. Studies were excluded if they were non-elective or transplant patients, non-English and not comparing ERAS to standard care.All potential studies and relevant data were retrieved and extracted by one author (LN). Data were extracted using a data extraction sheet agreed by all reviewers, and data extraction was subsequently validated by other authors. Data extracted included authors’ first and last names, study design, patient’ characteristics , type of surgery, outcomes measured, sample size, follow-up period and ERAS programme items.27 were excluded for the following reasons: 1 study included both hepatic and pancreatic surgery, 11 cohort studies did not include standard care, 4 RCTs did not include standard care, 1 paper related only to ERAS guidelines and 10 were reviews. The final 27 studies were included in the meta-analysis  and 1 point for power calculation , I2 statistic, with p < 0.1 considered to be statistically significant. A fixed effect model was used for pooling. Where there is significant evidence of heterogeneity (> 50%), random effect model was used instead. Using the method developed by devised Hozo, Djulbegovic and Hozo (2005), study data presented as medians and ranges or medians and interquartile ranges were converted to mean and standard deviation (SD). Funnel plots were used to assess presence of publication bias.This meta-analysis was performed using Review Manager (RevMan) version 5.3.35 16 were case-control studies42 and five prospective studies.34 Eleven studies included both open and laparoscopic surgery in the study,40 ten studies included only open liver surgery patients,42 five studies included only laparoscopic surgery patients,38 whilst one study performed a separate analysis for laparoscopic surgery and open surgery.21 A detailed characteristic of included studies is shown in Table A total of 3739 patients were included in the review (ranging between 26 and 347 per study), 1777 were managed according to an ERAS protocol and 1962 according to standard perioperative care. The ERAS elements utilized in the studies range between 8 and 23. A detailed list of ERAS elements utilized by each study is shown in Table Funnel plots for overall complication and readmission rates were used to assess publication bias Figs.  and 4. T41 three in Chinese yuan42 and two in euros.33 One study reported 30% reduction in hospital cost in ERAS group without providing further data,36 and one did not provide data on overall hospital cost.34 These two studies were therefore excluded from the meta-analysis.Twelve studies provided data on hospital cost (1606 patients). Five of the studies reported hospital cost in US dollar,p < 0·001), 21·5% in medical supply costs  and significant reduction in therapy-related costs (p < 0·001) were reported in the ERAS group, with no differences between the two groups in operating room or radiology costs. One study reported the hospital cost in mean without standard deviation.30 Using a method suggested by Furukawa et al.,44 standard deviation was borrowed from a study with similar sample size and mean.41A 40·7% reduction in laboratory-associated costs ($333), 54·1% in pharmacy-related costs . However, there was a significant heterogeneity observed among all studies . In the subgroup analysis for the type of studies, hospital cost was significantly lower in the ERAS group for both RCTs  and cohort studies . There was no significant difference in the overall hospital costs between the RCTs and cohort studies   and laparoscopic surgery  in the ERAS group over standard care. However, there was a significant difference in the overall hospital costs between the two surgical approaches , I2 = 95.6%) , with a significant heterogeneity observed in the studies . A subgroup analysis for RCT  and cohort studies  demonstrated a significant shorter LOS in the ERAS vs standard care group. However, there was a significant difference in overall LOS between RCTs and cohort studies   and laparoscopic surgery  in the ERAS group over standard care. There was no significant difference in overall LOS between open surgery and laparoscopic approach , I2 = 0%)  reported overall complication rates, with a total of 1332 patients experiencing complications (507 in ERAS group and 825 in standard care group). One study did not provide data on the number of cases of complicationp = < 0.00001) but with a significant heterogeneity observed in the studies . In the subgroup analysis, cases of overall complications were significantly less among the ERAS group than standard care in the RCTs , cohort studies . There was a significant difference between RCTs and cohort studies  . However, complication rates in ERAS group and standard care were similar for open surgery . There was no significant difference in the overall complication rates between the two surgical approaches , I2 = 0%). Moreover, Page et al.34 found significant fewer incidence of postoperative complications in the ERAS group  , with no significant heterogeneity observed in the studies . In the subgroup analysis, there was no significant difference in readmission rates between ERAS and standard care in both RCTs  and cohort studies . There is no significant difference in the overall readmission rates between RCTs and cohort studies   and laparoscopic surgery . There was no significant difference in the overall readmission rates between the two surgical approaches , I2 = 0%) . A total of 167 patients were readmitted (82 in ERAS group and 85 in standard care group). Pooling of the results, there was no significant difference in readmission rates between ERAS and standard care  Fig. . In a fu0%) Fig. .Fig. 11F39 A total of 20 deaths were reported in the studies (6 in ERAS and 14 in standard care group). Pooling the results, there was no significant difference in mortality rates between ERAS group and standard care , and no significant heterogeneity observed among the studies . Subgroup analysis for RCT  and cohort studies  found no difference in the mortality rates between ERAS group and standard care group. There was no difference in mortality between the RCTs and cohort studies  . Of these studies, no mortality occurred in ten.0%) Fig. .Fig. 13F40 Compliance was higher in the ERAS group across the three studies, ranging from 65% to 73.8% in the ERAS group and 20% to 48.7% in the standard group. Jones et al.25 reported 100% compliance in 18 out of the 19 ERAS elements in the ERAS group compared to 100% in 11 out the 19 ERAS elements in the standard care group.Four studies (474 patients) provided data on compliance to ERAS elements. Three of the four studies measured overall compliance to ERAS elements.46 we extracted sufficient data to conduct a subgroup analysis in hospital cost and confirmed a significant reduction in the overall cost following the implementation of ERAS protocol in liver surgery.This present review investigated the impact of ERAS programmes in liver surgery. A comprehensive search was performed resulting in 6 RCTs and 21 cohort studies with a total of 3739 patients, making this study the largest and most comprehensive review conducted on this topic to date. In addition to confirming that ERAS is safe and feasible, reduction of LOS and readmission rate without an increase in complications and mortalities in liver surgery,p < 0.0001). Whilst the main objective of ERAS protocol is not about healthcare cost savings, however, evident from this review confirms the results from two previous reviews10 that introduction of ERAS protocol in liver surgery represents a significant reduction in hospital expenses. It was also evident that any significant reduction in length of hospital stay and complication rates was accompanied by significant reduction in total hospital expenses following implementation ERAS programme.Regarding the primary outcome, hospital costs were significantly lower in ERAS group in every study included in the meta-analysis  including normal or decreasing serum bilirubin, good pain control with oral analgesia only, tolerance of solid food, no intravenous fluids, mobile independently or at the preoperative level and willingness to go home appear to be the most widely adopted by other liver units.Our findings suggested that LOS was significantly reduced by 2.22 days in the ERAS group, which is similar to previous findings of others.13 our findings again suggested a reduction in the overall complication rates in ERAS group compared to standard care, with a significant heterogeneity in the cohort studies. Contrary to the findings of Wang et al.,10 our results did not demonstrate a reduction in overall complication rates in open liver surgery. However, it is worth noting that this current review excluded studies that combined both minimal invasive and open surgical approach in the subgroup analysis. We believe that inclusion of these studies could introduce bias. Furthermore, our findings implied that complications in liver surgery are complex and cannot be reduced solely by ERAS pathway. Therefore, where possible, with the right technique and expertise, laparoscopic approach should be considered along with ERAS protocol to reduce risk of complications.Similar to previous reviews,42 suggesting Clavien-Dindo classification is the most common method of reporting and grading complications in liver surgery.Regarding method of assessing complications, eight of the cohort studies did not use Clavien-Dindo or mentioned the method by which postoperative complications were evaluated,p = 0.68 and p = 1.00, respectively). The reason for this could be explained by the fact that incidents of readmissions and mortalities reported in the studies were too low in the studies to detect any significant differences. For example, overall cases of readmissions were 0.06% both in ERAS group and standard care group . Similarly, cases of mortality were very low across the studies, with 6 deaths (6/1605) in ERAS group and 14 deaths in standard care group (14/1828). Follow-up was 30 days across the studies with the exemption of three studies that reported a 90 days follow-up.31 These 3 studies have a combined total of 458 patients (218 ERAS and 240 standard care), with 4 deaths (1 in ERAS group and 3 in standard care group). Similarly, two studies reported 90 days readmission.19 There was a 7.64% (11/144) readmission rates in ERAS group and 8.43% (14/166) in standard care group, respectively, which is similar to 5.9% (82/1387) overall average of readmission rates in ERAS group and 5.86% (82/1400) in standard care group, suggesting that 90 days follow-up may not be necessary.The cases of mortalities and readmissions were equally similar in the two groups  of the included studies reported performing a systematic audit40 to monitor of ERAS elements within the ERAS programme.There were significant variations in the number of elements utilized in each study. All studies applied 19 items or less with the exemption of Teixeira et al.One of the key limitations in this study was the presence of significant heterogeneity in the effect of ERAS protocols on LOS and hospital cost. The studies included in this review were conducted in 11 countries across 4 continents . Most of these countries have difference healthcare system which may have contributed to the significant heterogeneity observed in the analyses. One notable example was the wide variation in the hospital cost reported across the studies. Although, a random effect was used when there was a significant evidence of heterogeneity, there was no guarantee that heterogeneity can be eliminated impacting on the validity on our results. The majority of studies included in this review were conducted over a period of time when ERAS protocols were not standardized or fully established, with a wide variation in the elements applied in the protocols. Therefore, it is not clear whether this has introduced bias regarding the exact impact of ERAS protocols compared to standard care. Similarly, most of the included studies were based on retrospective data analysis introducing further bias to this review. Furthermore, the majority of the cohort studies were low-quality with small and unequal sample sizes which may lead to confounding bias. A larger high-quality multicentre and multinational RCTs is recommended to confirm some of the findings in this review.Our review concluded that the introduction of ERAS protocols is safe and feasible in hepatectomies, without increasing mortality and readmission rates, whilst reducing LOS and risk of complications, and with a significant hospital cost saving. Laparoscopic approach may be necessary to reduce complication rates in liver surgery. However, further studies are needed to investigate overall compliance to ERAS protocols and its impact on clinical outcomes.


Solanum lycopersicum L.) is a widely used model plant species for dissecting out the genomic bases of complex traits to thus provide an optimal platform for modern “-omics” studies and genome-guided breeding. Genome-wide association studies (GWAS) have become a preferred approach for screening large diverse populations and many traits. Here, we present GWAS analysis of a collection of 115 landraces and 11 vintage and modern cultivars. A total of 26 conventional descriptors, 40 traits obtained by digital phenotyping, the fruit content of six carotenoids recorded at the early ripening (breaker) and red-ripe stages and 21 climate-related variables were analyzed in the context of genetic diversity monitored in the 126 accessions. The data obtained from thorough phenotyping and the SNP diversity revealed by sequencing of ripe fruit transcripts of 120 of the tomato accessions were jointly analyzed to determine which genomic regions are implicated in the expressed phenotypic variation. This study reveals that the use of fruit RNA-Seq SNP diversity is effective not only for identification of genomic regions that underlie variation in fruit traits, but also of variation related to additional plant traits and adaptive responses to climate variation. These results allowed validation of our approach because different marker-trait associations mapped on chromosomal regions where other candidate genes for the same traits were previously reported. In addition, previously uncharacterized chromosomal regions were targeted as potentially involved in the expression of variable phenotypes, thus demonstrating that our tomato collection is a precious reservoir of diversity and an excellent tool for gene discovery.Tomato ( Solanum lycopersicum L.) is a major vegetable crop worldwide and a widely used model species in genomic studies and breeding. The rapidly increasing availability of genomic data has allowed researchers to bypass the limitations of a single reference genome and has facilitated development of a pan-genome that represents the genetic diversity of cultivated and wild tomato species [Tomato . In Sardinia in particular, together with artichoke, tomato is the most important horticultural crop species, with numerous landraces still cultivated locally by farmers. This is of special relevance if we consider that landraces might be directly used in marginal environments or exploited in future breeding programs as a reservoir of genes that underlie quality and productive traits that can be used to increase the performance of modern cultivars and to boost sustainable agriculture ,6,7,8,9.Genome-wide association studies have become a powerful tool to investigate and link the huge available amounts of genome sequence variation data with measurable phenotypic variations ,3,4. RecHere we used both conventional MLM and the two alternative models, FarmCPU and QTCAT, to scan an ad hoc assembled population of 120 tomato varieties that were mostly landraces for relevant associations among 2470 RNA-Seq–derived SNPs and traits of interest. In this “multi-omics” era, where DNA sequence data are easily acquired, phenotypic data are becoming the limiting factor. For this reason, we have here mainly addressed our efforts at thoroughly characterizing this population. Accordingly, the aim of the present study was to link the diversity observed at the phenotypic level with that observed at the transcribed genome level. To achieve this goal, we used three different GWAS approaches that provided an output of 536 significant associations related to plant and fruit morphophenological traits, fruit quality and response to climate. With the present study, we are acknowledging the value of this tomato collection for future gene-function, genome-editing and crop-breeding studies.Solanum lycopersicum L.) accessions that comprised 64 landraces from Sardinia, 8 landraces from other regions of Italy, 43 landraces from different countries and 11 vintage or modern cultivars  during 2012 and 2013. We conducted the open-field trial in 2012 at Oristano, according to a randomized complete block design with five replicates, 124 treatments (accessions) and four plants per plot. The field trial started in June and the full mature fruit were harvested in September. In 2013, we set up the second trial from January to July in a greenhouse in Ottava (Sassari); a randomized complete block design was again used, with three replicates, 126 treatments (accessions) and three plants per plot.http://tinyurl.com/n7k75m6).We used 26 conventional morphophenological traits  to charaIn addition to these conventional descriptors, we carried out digital phenotyping on the mature fruit collected in 2013 using the Tomato Analyzer software ,25. For We extracted the carotenoids from three replicates of each accession (one per block), at both the breaker stage , when the fruit are still rich in chloroplasts and the red-ripe stage, when the fruit accumulate large quantities of carotenoids, for a total of 762 samples. All of the harvested fruit at the breaker and red-ripe stages were healthy and uniformly colored within their stage. An example of the two ripening stages is shown in Frozen flesh samples from each fruit stage were rapidly homogenized and carotenoids extraction performed as described in . Carotenhttp://www.diva-gis.org/) was used to extract ecological data from the free-access database at http://www.diva-gis.org/climate.For each accession for which geographic coordinates were available (108 accessions), climatic data were also downloaded relative to each collection site. Bioclimatic variables were used for further comparative analyses with phenotypic and molecular data, as they are considered more biologically meaningful than monthly temperatures and rainfall. Indeed, they describe annual temperature and precipitation trends, seasonality or extreme environmental factors . The DIVA-GIS 7.5 software (http://broadinstitute.github.io/picard/) and only uniquely mapped reads were kept. SNPs were then identified based on the mpileup files generated by SAMtools [Transcriptome sequencing was performed on 120 of the 126 accessions cultivated during the green-house trial in 2013. The fruits used for RNA extraction were the same used for carotenoid extraction (see above) Total RNA was extracted using RNeasy Plant Mini kits  from full-ripe tissue samples previously frozen at −80 °C and homogenized. Strand-specific RNA-Seq libraries were constructed using a protocol described in  and 51-bSAMtools . The resijr is the observation of the ith genotype, in the jth environment and block replicate r, μ is the grand mean, Gi is the effect of the ith genotype, Ej is the effect of the jth environment, G*Eij is the interaction of the ith genotype with the jth environment, Br(Ej) is the effect of the rth replicate in the jth year and εijr is the random error.Ranges and mean values were calculated by site for each accession on quantitative conventional descriptors, Tomato Analyzer descriptors and carotenoid contents. Analysis of variance (ANOVA) was then performed on the conventional descriptors collected from the 122 accessions shared among the two sites. To test for significant variations among environments and genotypes and to estimate the size of the genotype by environment interaction (GxE) we used the model given in Equation (1):The adjusted means (as the best linear unbiased predictors (BLUPs)) and the broad sense heritability were also calculated across sites for the quantitative conventional descriptors and within sites for the Tomato analyzer descriptors and the carotenoid contents. To calculate these statistics, we have fitted a mixed linear model based on the restricted maximum likelihood method with years as fixed effects and genotypes and blocks as random effects using Equation (2): 2p = σ2g + (σ2g⋅e/ne) + (σ2ϵ/ne⋅nr) for heritability across the two sites and σ2p = σ2g + (σ2ϵ/ne⋅nr) for heritability calculated by site, and where σ2g is the genotypic variance, σ2p is the phenotypic variance, σ2g⋅e is the genotype by environment interaction, ne is the number of environments, σ2ϵ is the residual variance and nr is the number of replicates. The expression given in Equation (3) was used to calculate the statistics:lme4 and car) [All the above statistical analyses were carried out using R dedicated packages (and car) .pi is the frequency proportion of the descriptor state and b is the base of the logarithm. Most studies use natural logarithms, although some use base 2 (which makes no significant differences). Each value was normalized by dividing it by its maximum value  to keep the values between 0 and 1. Pearson’s correlations were estimated among the different traits using the cor standard R function and plotted using the corrplot package [ggbiplot package (https://github.com/vqv/ggbiplot). The Hopkins statistic [factoextra R package (https://cran.r-project.org/web/packages/factoextra/index.html).The Shannon–Weaver index was used to evaluate the diversity among the qualitative traits . GenAlEx package . Principtatistic  was usedtatistic . The EucST values and also proportion of shared alleles between pairs of sub-populations.Genetic diversity analyses were performed using GenAlEx 6.5  and Arlemantel.rtest function of the ade4 R package; 9999 permutations were used to evaluate the significance of the test. The phenotypic distances were those obtained to draw the clustering plot, while the genetic Nei minimum distances were obtained using the R adegenet [popr packages . A Mantel correlation test was then used to compare the genetic and phenotypic distances using the adegenet  and popradegenet package for the R software [2 < 0.2). To filter the dataset, we used the R package bigsnpr [To investigate population structure, we used the model-based clustering method, as implemented in Structure vs. 2.3.4 , and dissoftware . The firsoftware . The secsoftware . The met bigsnpr , to obta2 measure was calculated; in the second case, the r2 unbiased estimates were corrected by the structure of the sample (rs2), the relatedness of the individuals (rv2) or both (rvs2). These methods were implemented in the LDcorSV R package [LD-vignette included in the Bioconductor package snpStats implemented in R [2 data against the genetic distance and fitted the LD decay line as in [https://fabiomarroni.wordpress.com/). The regression line is based on [SneP, a program designed to easily estimate effective population size from genome-wide SNP data or directly from LD levels [To further evaluate the structure of the population, we estimated the familial relatedness and linkage disequilibrium (LD) levels of the tomato collection by calculating the pairwise kinship coefficients as obtained from TASSEL 5.2.42 . We then package . Pairwisted in R ,46. We tne as in , adaptinbased on  and the D levels .2 value) across our tomato population above which loci can be assumed to be associated, we used the method from [To identify the critical value of LD , genotype (G) and genotype × year (G × Y) interactions as effects of the model revealed significant differences among the genotypes between the years, except for FWG, NFI and LWI (p < 0.001) was also observed for G and G × Y interaction effects (2B) calculated across sites and by site also varied among conventional traits  differences were observed in both 2012 and 2013 when looking at the differences among the genotypes, as shown by the mean, maximum and minimum values of the conventional quantitative traits  . Almost 2B = 92.21%) and the lowest for the perimeter of the longitudinal section (h2B = 36.65%)  . The meaFor the carotenoids, the total content was higher at the ripe stage than the breaker stage, as expected . At the The heritability of the carotenoids was on average lower than the other phenotypic traits, with β-carotene showing the highest values at both breaker and ripe stages  .p < 0.0001) with the area, perimeter and maximum width (Width_M_H_L and Width_M_H_T) of the transverse and longitudinal sections of the fruit . Significant correlations were also observed between carotenoid content and fruit color and size. In particular, β-carotene content was negatively correlated  to the mean fruit weight (FWG) at both the breaker stage and the ripe stage and it was positively correlated with the degrees Brix at the breaker stage  and the ripe stage . Fewer correlations were detected between bioclimatic variables and the plant/fruit traits, some of which were of particular note, such as those between the traits that described fruit size  and bio3 , bio8 (Mean Temperature of Wettest Quarter), bio12 , bio13 (Precipitation of Wettest Month), bio 16 (Precipitation of Wettest Quarter) and bio18 (Precipitation of Warmest Quarter). The observed correlations varied from −0.45 (p < 0.001) for correlations between FLE and bio3 and bio8, to −0.27 (p < 0.005) for correlations between area and perimeter with bio12, bio13 and bio16  and low puffiness. Cluster C is mainly comprised of varieties of small/medium size, elongated fruit, with thick pericarp and intermediate phenotypes for sugar content.The main features of the three groups can be deduced from the loading of the different traits on the first two principal components . BrieflyST) among the groups was 0.07 on average and was always statistically significant. Moreover, the proportion of shared alleles indicated higher similarity between SLR and ELR than between CV and ELR (The genetic differentiation (F and ELR . On the  and ELR .p < 0.001) among populations. This thus indicates that more than 96% of this diversity is due to the genetic variation among accessions within groups, either from different countries of the world or from a single country or region .The partitioning of the SNP variance (AMOVA) indicated that only 4% of the total variation was due to a statistically significant differentiation . Indeed, if we do not consider the admixed individuals, the same accessions were attributed to the green and red genetic groups shown in Based on the Bayesian approach, the most likely number of genetic groups within this tomato population was three , while tp = 2 × 10−4; 9999 permutations), the genetic and phenotypic clustering do not completely overlap  was higher than the r2 measure corrected for population structure (rs2), kinship (rv2) and both population structure and kinship (rvs2). These were seen as r2 > rs2 > rv2 > rvs2; rv2 and rvs2 LD were very similar overall and within each chromosome  and rv2 (1.37 Mb) than for standard r2 (1.06 Mb). This output was more evident within chromosomes for the rs2 decay.We plotted the rromosome  and the Some differences were also observed for LD decay among the different chromosomes, with lower LD decay distances for chromosomes 1, 3, 8 and 10, while the highest decay distances were for chromosome 5  that encode transcription factors  were detected within 203 genes, 23 of which are genes  was detected for fruit size and shape  , which was detected by the three GWA models as associated to several traits  and was located within the gene Solyc02g081700, which encodes a proteasome subunit α type protein, 13 kb from an ANANTHA gene  is located within the gene it size) . This geTHA gene .Recruiting Motif (TRM) gene that has been previously shown to influence final fruit shape [Among the genomic regions that influenced variation of fruit traits, an MTA on chromosome 4 (ch4_31) was 28 kb from a TONNEAU1 it shape .FASCIATED gene is located. FASCIATED has been extensively studied in tomato, where mutations to this gene result in altered floral meristem size and locule number [On chromosomes 6 to 10, we detected more relevant MTAs for fruit size and shape, of which six were located within genes that encode transcription factors and others were either located near genes that influence fruit setting  or in regions where no genes determinant for the investigated fruit traits were found . On chroe number ,56,57,58Solyc01g079620, a SlMYB12 gene that influences accumulation of the yellow-colored flavonoid  in tomato fruit [Among the associations detected for quality traits, we found a relevant region on chromosome 1 (at 71 Mb) where three SNPs (ch1_38-40) were associated to fruit color and β-carotene content . These Sto fruit . These MSolyc02g081700, above mentioned for its association with different fruit size and shape traits and one located within Solyc06g066320, the gene encoding for a transcription factor homolog of Arabidopsis thaliana IWS1 (INTERACTS WITH SPT6) [Other relevant MTAs were detected for sugar content and other fruit quality traits. Among these, one is located within the gene TH SPT6) . This geSolyc01g108020, a gene that encodes a thioredoxin M3, and one on chromosome 10 within Solyc10g084400, which encodes a glutathione S-transferase (AP2/ERF). Moreover, on chromosome 11 we detected a SNP (ch11_49) within a DELLA gene (Solyc11g011260) that encodes a gibberellic acid insensitive (GAI) transcription factor  within nsferase . These Mn factor . The gend tomato ,63,64,65Solyc04g080730, at 31 kb from Solyc04g080780 and 35 kb from Solyc04g080790, two BEL-like homeodomain genes [Arabidopsis and tomato [Solyc11g010490 at 82 kb from the J-1 gene, a MADS-box 512 transcription factor implicated in the differentiation of the pedicel abscission zone and the 513 maintenance of the inflorescence meristem [Solyc11g071680, which encodes a serine/threonine kinase (homolog of the Arabidopsis TOUSLED). This is particular interesting because it encodes a serine/threonine protein kinase that is required for leaf and flower development and is involved in the regulation of RNA interference [Marker-trait associations for leaf size were also identified that included an MTA for leaf length within in genes . BEL-LIKd tomato ,67,68. Mmeristem ,70 and arference . These fSolyc02g071510, a transcription factor GTE12, which is associated with bio13, bio15 and bio16 . The T allele of ch2_130 is private to the exotic landraces group  and were both identified within  protein . This pr LOCUS C ,74.Solyc05g015510 that it is associated with precipitation  and encodes SlySBP10 a SQUAMOSA promoter-binding protein  by activating a central flowering repressor that is encoded by flowering locus C [Solyc07g055660, located 26 kb from a heat-stress transcription factor; Solyc08g076930, a transcription factor for jasmonic acid 3 that is 72 kb from a RAMOSA transcription factor  to be a worldwide sample. On the other hand, the higher levels of genetic diversity observed within modern and vintage cultivars underlines that the breeding programs might have led to inclusion of different loci in these accessions ,16.Previous studies performed on tomato landraces and commercial cultivars using SNP markers revealed comparable levels of diversity and similar levels of heterozigosity when looking at groups of landraces from different regions ,81,82,83The genetic distance observed among the groups of varieties was not high, which is consistent with the nature of domesticated tomato, for which high phenotypic but low genetic variation has been shown previously ,11,16,85The population structure and the linkage disequilibrium analyses performed prior to GWAS have further shown that our population was adequate for association analysis. While the LD levels and LD decay on some chromosomes were comparable to those from previous studies, these were quite variable among chromosomes ,82,87. IAmong the methods used to perform these GWAS, FarmCPU and QTCAT shared the highest numbers of associations while the standard MLM method, showed the least number of shared associations with the other two methods. These data indicate that some associations are highly robust and detected by all methods and that weaker associations might best be identified using multiple strategies. In particular we also chose to use QTCAT to determine whether the associations detected were comparable with those from previous consolidated GWAS models ,19,20,21Differently from many GWA studies that rely on the genomic variation, the present study was based on the transcriptome variation among genes and accessions. The RNAseq based SNPs genotyping has been proven to be valuable for QTL mapping and functional and evolutionary studies ,93. ConcThe GWAS results have mainly targeted genomic regions that underlie genes relevant for fruit size and shape traits. This is a direct consequence of our phenotypic analysis, which mainly focused on fruit traits, in line with the relatively thorough phenotypic characterization, as either detected by conventional or digital phenotyping. This is also a good way to evaluate the appropriateness of the present collection for GWAS studies. Indeed, a large number of associations were detected with data from the Tomato Analyzer, which was also successfully used in previous studies ,86,100. Arabidopsis CLAVATA3), which underlies the fasciated mutant phenotype [Additional traits identified by these authors as predictive for these categories were fruit shape index, distal end protrusion, width widest position, proximal end blockiness, rectangular, distal angle and proximal eccentricity. Here, we also detected many of these associations, such as that with Proximal_angle_macro near OVATE or with fruit-shape-related traits near FASCIATED. However, interestingly, along with these associations, we also detected further associations linked to SlCLV3 . These show that this combined regional and global tomato collection is useful for targeting genes with prominent roles in fruit. As an example, a single SNP (ch2_151) was identified within a gene that encodes a proteasome subunit α type protein that appears to have a key role during fruit setting ,108. ThiTogether, these results suggest that the diversity of this germplasm is sufficient to gain further insights into the role and function of genes that underlie important traits. Indeed, further associations were found in regions that were previously targeted as important for fruit color, carotenoids and sugar content, while others targeted novel genes that might be of relevance for quality traits, such as fruit color and β-carotene content on chromosomes 1 and 4 ,111,112.Using GWAS on SNPs from RNA-Seq extracted from mature fruit, we did not expect a direct implication for most of the present polymorphisms in the expressions of genes involved in plant development and phenology. However, we would expect a relevant number of these genes to be in LD  with others expressed in the fruit tissue. The significant RNA variation has indeed allowed the identification of associations for growth habit. These were often at short distances from transcription factors that have determinant roles in the regulation of plant growth and development, including tomato ripening and responses to environmental stimuli ,114,115.Solyc06g066320, the gene encoding for a IWS1 transcription factor, is particularly interesting because this protein is highly conserved in different organisms (from humans to yeast) in Arabidopsis it is required for gene expression induced by brassinosteroids [gene spanning nearly 0.5 Mb, is enriched of genes encoding for different transcription factors, also related to fruit development and ripening [Arabidopsis, as well as in the establishment of polarity of lateral organs, including leaf and flower, in Arabidopisis, rice and other species [Solyc06g066320, together with its simultaneous associations with different traits , suggest a possible pleiotropic role for this gene. Being pleiotropy a target for many GWA studies, variable strategies have been designed for the identification of causal variants and their roles that will help to extract the huge amount of information that is being produced [As revealed by a recent study from Wu and colleagues , genes osteroids ,116. Morripening ,117. In  species ,119,120.produced ,121,122.Previous studies based on climatic data have revealed gene expressed regions that might be involved in the answer to different environments ,127,128.Solyc02g071510, which encodes a transcription factor (GTE12) that in Arabidopsis is implicated in the cytosolic Ca2+ increase after application of stress, such as heat, drought and cold [Among the most interesting results, we found MTAs identified on chromosome 2 for variables related to precipitation, such as ch2_130, with its “T” allele being private to the exotic landrace population. This MTA is located within and cold ,130. Thrand cold ,128,131.Further associations were detected across the remaining chromosomes in genomic regions that appear to be involved in plant responses to climatic changes. As an example, we indicate the MTAs located within a gene that encodes a heat stress transcription factor or within SlMYC2, a jasmonic acid transcription factor involved in fruit chilling tolerance in tomato . Others The present results indicate that we can select the accessions that carry the polymorphic alleles to create specific crosses with controlled fruit size and shape and, for example, improved quality characteristics or that might be linked to specific climate responses.We demonstrate here the value of exploring the genetic diversity of local and regional accessions, to exploit the variation of this genetic diversity reservoir to gain knowledge and facilitating crop improvements. Our results indicate that, as well as the relevant associations detected for fruit size, shape and quality, there is correlation between the expression of fruit traits and other traits related to plant development and flowering, and the likely response to climate variations. As recently demonstrated in tomato, the human-driven selection for specific traits during domestication has resulted in direct effects on other secondary traits or indirect influences in the expression of other genes to linkage drag ,133. Som


Two independent cohorts were prospectively collected, cohort A , cohort B . DNA was extracted and its integrity evaluated with a qPCR-based assay that produces a normalized integrity index, the QC score (ratio between the quantity of a long and a short amplicon of the same gene). We observed higher QC scores in coldFFPE compared to stdFFPE samples  and stdFFPE breast cancer specimens showed the most detrimental effect overall. Comparable QC scores were obtained between coldFFPE tissues of both cohorts; conversely, DNA integrity of stdFFPE was significantly lower in cohort A compared to cohort B (p < 0.0001). Of note, QC scores of stdFFPE (but not of coldFFPE) samples were significantly reduced following 6 months of storage (p = 0.0001). Monitored formalin fixation at 4°C outperforms standard fixation in ensuring high-quality DNA, which is key to feasibility of downstream high-throughput molecular analyses. An important effect was observed over storage time, thus suggesting a likely better preservation of archival samples when this cold fixation protocol is used.Formalin fixation and paraffin embedding (FFPE) represent the standard method to preserve tissue specimens for diagnostic pathology, however formalin fixation induces severe fragmentation of nucleic acids. We investigated whether formalin fixation at 4°C could preserve DNA integrity in FFPE specimens. Paired samples from 38 specimens were formalin fixed at room temperature ( The harmonization of pre-analytic procedures represents the corner stone of optimal diagnosis in pathology , 2. The It is widely accepted that formalin fixation exerts a blasting effect on both DNA and RNA, with damages comprising fragmentation, non-canonical cross-linkage and base alterations, with critical proportional consequences related with storage time , 4. Of nWe have previously reported on a protocol, i.e., cold formalin fixation, which improves RNA quality without interfering with the formalin ability to preserve both tissue structure and antigen reliability for immunohistochemical analyses . This moBased on these premises, the aim of our study was to comprehensively characterize a series of DNA samples purified from tissues derived from the same surgical resection but processed with two different fixation protocols (standard fixation vs. cold fixation at 4°C), and to assess whether results would differ based on the tissue of origin. Fluorometric, spectrophotometric and qPCR-based methods were applied to assess DNA integrity.ad hoc collected from surgical specimens handled by under-vacuum packing and cooling (VPAC), as previously described (standard fixation procedure (stdFPPE): samples were fixed for 24 h in 4% neutral-buffered formalin (NBF) at room temperature (RT). Subsequently, samples were processed to paraffin embedding with an automatic tissue processor and embedded in paraffin wax; (ii) cold fixation procedure (coldFFPE): the sample was immersed in pre-cooled 4% NBF and fixed at 4°C for 24 h. For this protocol, specimens were dehydrated in ethanol 95% at 4°C for 4 h and then followed the same stdFFPE sample processing without the first ethanol 95% step by using the automated tissue processor. All of the methods were performed in accordance with the relevant guidelines and regulations.The 38 paired samples included in the study were escribed , 9, 10. escribed . Of noteOut of the 38 cases, 21 pairs were collected and fixed 6 years prior to the present study  and 17 samples prospectively collected at time of the present study  . Cohort stdFFPE and coldFFPE samples. Data were analyzed anonymously. Five 8-μm thick sections were dissected and DNA samples were purified from all the 38 FFPE pairs for a total of 72 DNA samples. In addition, in 14 cases from cohort B for which at least 6 months elapsed from collection paired stdFFPE and coldFFPE samples underwent a second DNA extraction 6 months after collection/fixation.A pathologist evaluated histological and pathological features of the cases included in both cohorts and the tumor area was identified on the hematoxylin and eosin (H&E) stained slide before proceeding to the experimental procedure for both DNA was extracted using the QIAamp DNA FFPE Tissue Kit  according to the manufacturer's protocol, following an overnight 56°C tissues lyses allowing a complete tissue digestion without fragmenting the DNA. DNA was eluted in 40 μL of nuclease-free water and quantified using the Qubit 3.0 Fluorimeter  following the protocol of High Sensitivity DNA Kit . To check for any possible contaminant on the purified samples, DNA concentration was also evaluated using the DeNovix DS-11 UV-Vis Spectrophotometer  to obtain the 260/230 and the 260/280 nm ratios.DNA fragmentation was evaluated by using the DEPArray™ FFPE QC Kit . This qPCR-based kit is a multiplex reaction composed of two primers pairs flanking two regions of 54 and 132 bp of the same genomic locus. To quantify the amount of the produced amplicons, a standard curve for each primer pair was generated. To infer the DNA integrity level, the ratio between the quantity of the long amplicon  and the short amplicon is calculated. This ratio, named QC score, is a normalized number tending either to 1, in a context of highly conserved DNA, or to 0, in a scenario of diffuse DNA degradation.R2 parameters of the standard curve may be between 3.1 and 3.6 and 0.99, respectively. The fluorescence unit threshold was set at 0.04 for all the experiment.Briefly, the standard curve points were produced with serial 1:10 dilutions from an undiluted standard with a known concentration of 20,000 pg/μL. To perform the assay, 8 μL of DNA with a concentration of 2 ng/ μL were added in two mix composed by 10 μL of the Master Mix and 2 μL of the Short or Long Primer mix for a final volume of 20 μL. The thermal cycle was run on a RotorGene Q instrument (Qiagen) and contemplated an initial step of DNA denaturation for 10 min at 95°C followed by 40 cycles of 20 s at 95°C denaturation and 1 min at 57.5°C annealing/extension with the detection of FAM fluorescence signal at 520 nM. To obtain optimal results, slope and (CTsample-INTERCETstdcurve/SLOPEstdcurve) for both the short and long amplicon. The QC score was obtained by dividing the long amplicon RQ by the short amplicon RQ.All samples and standards were analyzed with technical triplicates in each experiment. The amount of amplified DNA was checked analyzing the quantity of the short and long amplicons assay products. By comparing the average Ct of the three replicates with the standard curve the relative quantification (RQ) of PCR product was obtained using the formula 10stdFFPE and coldFFPE samples to determine any intra-specimen influences.Statistical analyses were carried out using GraphPad Prism statistical software v8.0 . After normality test, we applied both the unpaired and paired distribution test to evaluate significant differences in QC scores between the two-fixation methods. The unpaired distribution test was used to assess differences by considering the QC scores as independent measurement of uncorrelated samples, whereas the paired distribution test evaluated the statistical distribution considering the different fixation of the same sample as repeated and correlated data. The Pearson's correlation coefficient test (r) was used to determine the correlation between QC scores of corresponding p-values of <0.05, with 95% confidence intervals (CI), were considered as statistically significant.We first analyzed the total cohort by merging the data obtained from the two sets and then by separately considering cohort A and cohort B. stdFPPE and coldFFPE samples were evaluated by pathologists blinded to the experimental procedures of fixation. Only tumor cell areas were selected for dissection and DNA purification. Tumor cell composition was comparable between corresponding samples for each case; minor differences were observed in terms of relative percentage of immune cells populating the intertumoral stroma. Necrotic areas were carefully avoided through mesodissection. Only two sample pairs were characterized by large necrotic areas interspersed among tumor cell clusters and most likely contained a non-negligible degree of necrosis in the extracted material, nevertheless no evident technical-derived alterations were detected. Of note, the pathologists involved in the assessment of morphological features did not raise observations on fixation artifacts in any of the samples.The H&E sections of corresponding To determine any influence of the different fixation protocol on DNA quantity and quality, we both applied fluorometric and spectrophotometric methods to evaluate the DNA amount. DNA concentration and ratios are reported in p = 0.2) were detected by comparing these data with the DNA quantity obtained from coldFFPE samples, which were slightly higher by the fluorometric  and spectrophotometric measurement .From the standard fixed samples, we purified a mean level of 4.2 (range: 0.2–15.6 μg) and 16.2 μg (1.9–57.7 μg) of DNA analyzed with Qubit or DeNovix instruments, respectively. No significant differences  using the Qubit fluorometer instrument, which was not appreciated in Cohort B. For both fixation methods the nucleic acid yield was significantly higher in Cohort B than in Cohort A , however the difference was less evident when comparing coldFFPE than stdFFPE specimens (40% of reduction against 70%)  .No correlation between DNA amount and type of tumors was detected. Finally, the fixation protocol did not influence the DNA quality, defined as the absorbance ratio between 230/260 and 260/280 nm, which resulted comparable between the pairs.R2 scores of the standard curves were 0.994 and 0.993 for the short and the long amplicon, respectively, and the line slopes were comprised in the optimal range defined by the manufacturer's protocol.The qPCR-based DEPArray™ FFPE QC Kit returned a QC score for each sample, a normalized evaluation of DNA amplifiability. From a technical standpoint, the mean stdFFPE DNA samples were characterized by mean QC score of 0.36 (range: 0.03–0.86), which was significantly lower than the corresponding value in coldFFPE samples . By comparing the QC score distributions of the two sets of samples, both the paired and unpaired tests reached the statistical significance (p < 0.0001) (r = 0.45)  . We wond = 0.45) .coldFFPE samples were substantially comparable between the two cohorts, regardless of time of fixation (p = 0.79), whereas the QC score of stdFFPE samples was significantly lower in Cohort A compared to the more recent prospectively accrued samples of Cohort B (p < 0.0001) , in contrast with the corresponding coldFFPE samples that showed a 6-fold increase in the QC average . On the other hand, the DNA purified from the 17 paired samples of Cohort B displayed a relatively better preservation in both stdFFPE and coldFFPE, nevertheless coldFFPE samples still showed a statistically significantly higher QC distribution  we observed that the QC scores of  0.0001) . Within stdFFPE breast carcinoma samples, and these values were significantly lower compared to all the other samples, either stdFFPE or coldFFPE (p = 0.0009). By focusing on the same samples treated with cold formalin, breast specimens confirmed the tissue-specific DNA degradation, compared with lung and colon cancer tissues (p = 0.02), more pronounced within the standard fixed samples (p = 0.0002). By investigating the intrasample QC score ratio between stdFFPE and coldFFPE, the highest advantage from cold formalin fixation was observed for DNAs purified from breast carcinoma specimens, with average 7.7-fold increase in the long amplicon in the coldFFPE compared to the stdFFPE samples  and comparable QC scores for the coldFFPE samples  (Based on the lowest level of DNA integrity observed in llection . We obses of 9%) .coldFFPE as compared to those from stdFFPE tissue samples. In addition, the results in our hands suggest an important impact of time of storage of FFPE samples on DNA integrity that can be circumvented by cold fixation. This seems to be particularly important for breast cancer specimens, which showed the most detrimental effect with standard fixation overall.Our experimental study shows that formalin fixation of tissue specimens at 4°C (cold fixation) leads to a better preservation of DNA as exemplified by the significantly lower levels of fragmentation in DNA samples obtained from BRCA1 and BRCA2 single nucleotide variants and copy number variations that can address patient to targeted therapeutic approaches  to the diagnostic process, leading to either a better definition of the lesion, or a better prognostic and/or predictive stratification of the disease (precision medicine). For instance, depending on the site of origin and on the histologic type of a tumoral lesion genetic alterations such as mutations, translocations and gene expression signatures are currently being assayed in clinical practice [reviewed in ]. As poiproaches , are strproaches , 15. Theproaches .coldFFPE samples, which showed significantly higher QC score values compared to stdFFPE DNA samples. Of note, the degree of statistically significant difference between coldFFPE and to stdFFPE DNA samples was higher in Cohort A, composed of specimens collected 6 years prior to the present study and whose DNA was recovered at time of the present study, compared to the Cohort B, composed of samples collected and extracted at present time. It is important to note that QC scores of the coldFFPE were comparable between the two cohorts, thus demonstrating consistency in obtaining high quality DNA from coldFFPE samples, whereas we observed relatively higher QC scores in stdFFPE DNA samples belonging to the prospective cohort, which rendered the difference with coldFFPE samples less evident, even though still statistically significant.Cold formalin fixation has been used in other studies, mainly focused on RNA integrity. Our group  and othestdFFPE samples compared to the first extraction, whereas comparable results were obtained between first and second DNA extraction for coldFFPE samples, hence strongly suggesting a better preservation of DNA in tissue specimens fixed in cold formalin. As a possible mechanism contributing to this phenomenon one could hypothesize that cold formalin fixation may be able to reduce both the first enzymatic degradation of DNA by blocking the DNAse activity  demonstrating that cold fixation in core biopsies of breast carcinomas results in optimal morphology and reliable protein detection by immunohistochemistry-based assays. Further studies are warranted to ascertain whether the advantage in terms of DNA integrity demonstrated for FFPE samples of surgical specimens is also observed in core biopsy and cytology samples, which are smaller in size and may undergo a shorter duration of formalin fixation.Despite these limitations, the precise quantification of the degree of DNA fragmentation that we performed with a normalized evaluation of the QC score revealed a clear benefit for DNA preservation in samples fixed in cold (4°C) formalin. Hence, we here provide a method to limit DNA degradation that is likely to (i) ensure a better performance of molecular diagnostic tests, (ii) enable a higher complexity of genomic analysis on DNA extracted from FFPE samples, (iii) anticipate a paradigm shift in pathology laboratories with the creation of FFPE archival samples that may be better preserved over time and foster therefore a higher accuracy and throughput of predictive molecular pathology assays, which have the potential to ultimately impact on the quality of life of breast cancer patients in the era of precision medicine.The datasets generated for this study are available in the The studies involving human participants were reviewed and approved Ethical Committee of Fondazione Piemontese per l'Oncologia- Istituto di Ricerca e Cura a Carattere Scientifico of Candiolo, CANDIOLO, Prot. Profiling 001-IRCC-00IIS-10. All subjects gave written informed consent in accordance with the Declaration of Helsinki.CM and AS: conceptualization and funding acquisition. EB, LA, UM, CP, EP, EM, DB, PC, AP, IS, TV, and CM: methodology. EB, CM, and AS: formal analysis and investigation. EB and CM: writing—original draft preparation. CM, AS, LA, and TV: writing—review and editing.CM has received personal/consultancy fees from Axiom Healthcare Strategies, Daiichi-Sankyo, MSD, Roche and Bayer, outside the scope of the submitted work. The remaining 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 handling editor declared a past co-authorship with one of the authors CM.