Alzheimer’s disease (AD) is a neurodegenerative disorder representing the major cause of dementia. It is characterized by memory loss, and cognitive and behavioral decline. In particular, the hallmarks of the pathology are amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs), formed by aggregated hyperphosphorylated tau protein. Oxidative stress plays a main role in AD, and it is involved in initiation and progression of AD. It is well known that Aβ induced oxidative stress, promoting reactive oxygen species (ROS) production and consequently lipid peroxidation, protein oxidation, tau hyperphosphorylation, results in toxic effects on synapses and neurons. In turn, oxidative stress can increase Aβ production. For these reasons, the administration of an antioxidant therapy in AD patients was suggested. The term vitamin E includes different fat-soluble compounds, divided into tocopherols and tocotrienols, that possess antioxidant action. α-Tocopherol is the most studied, but some studies suggested that tocotrienols may have different health promoting capacities. In this review, we focused our attention on the effects of vitamin E supplementation in AD animal models and AD patients or older population. Experimental models showed that vitamin E supplementation, by decreasing oxidative stress, may be a good strategy to improve cognitive and memory deficits. Furthermore, the combination of vitamin E with other antioxidant or anti-inflammatory compounds may increase its efficacy. However, even if some trials have evidenced some benefits, the effects of vitamin E in AD patients are still under debate. APOE gene can influence AD susceptibility [Alzheimer’s disease (AD) is a fast, progressive neurodegenerative disorder and represents the major cause of dementia in the elderly. The clinical features are memory loss, and cognitive and behavioral impairment . The haltibility .695, APP751 and APP770, which are generated by alternative splicing of exons 7 and 8. In the brain, the APP695 isoform is mainly expressed [1–40 is the most abundant form of Aβ in the brain, but Aβ1–42 is the more toxic due to its tendency to aggregate and form oligomers, and it is the Aβ isoform associated with AD [1–40, Aβ1–42 and other components [1–42 oligomers, whose levels correlate with the severity of the cognitive decline in AD [1–42 caused a marked increase in oxidative stress markers compared to fibrillar Aβ1–42, and, as a consequence of oxidative stress, also increased cell death [Aβ is formed by the cleavage of the APP, a type I membrane glycoprotein. However, APP is not simply the Aβ precursor, but it participates in normal brain functioning. Indeed, even if its role is not completely clear, it is involved in brain development, memory and synaptic plasticity. Three isoforms of APP exist, namely APPxpressed ,7. The βxpressed ,7. Aβ1–4 with AD ,11. Aftemponents . At firsne in AD ,14. Furtll death .The alteration of Aβ brain level in AD may be caused by a variation in its production, but also of its clearance. The clearance is mediated by different pathways, including the activation of degrading enzymes and receptor-mediated cellular and vascular clearance. Among the receptor, low-density lipoprotein receptor related protein 1 (LRP1) mediated clearance of Aβ. Also, P-glycoprotein is involved in Aβ clearance. Aβ is degraded by some peptidases, including neprilysin, insulin-degrading enzyme (IDE) and endothelin-converting enzyme .2O2), lipid peroxides, protein oxidation and oxidative stress markers [1–42 oligomers can insert into the lipid membrane forming α-helices and start reactive oxygen species (ROS) production, causing lipid peroxidation and protein oxidation [N-methyl-d-aspartate receptor (NMDA-R) activation [Oxidative stress represents one of the main factors involved in AD initiation and progression . Indeed, markers ,16,17. Ixidation . ROS proxidation ,19. De Fxidation  found thtivation .In turn, oxidative stress can enhance Aβ production. Indeed, mitochondrial dysfunction, enhancing ROS production, induced high levels of Aβ . Moreove1–42 oligomers promote tau hyperphosphorylation, resulting in toxic effects on synapses [In addition, it seems that Aβ induced acceleration of tau pathology and about this it has been observed that Aβsynapses ,23. The synapses .The term vitamin E refers to different fat-soluble compounds present in plants, that can be divided in tocopherols and tocotrienols, that may be differentiated for the presence of the unsaturated side-chain, presenting three double bonds. Each class has four homologs and the eight members are distinguished as α-, β-, γ-, and δ-tocopherol, and as α-, β-, γ-, and δ-tocotrienol based on the number and localization of methyl groups .Vitamin E is an essential micronutrient for humans, useful for maintaining the integrity of cell membranes. The daily dietary intake recommended for vitamin E vary between 3 and 15 mg in different countries and varies depending on age . Seeds aThe main vitamin E role is its antioxidant action, given that it is the most important lipophilic radical scavenging antioxidant in vivo. Vitamin E scavenges free radicals mainly by hydrogen atom transfer reaction obtaining a non-radical product and a vitamin E radical, that may react with another radical to give a stable product, attack lipids, or react with a reducing agent such as vitamin C or ubiquinol to regenerate vitamin E . α-tocopVitamin E is an important factor for other processes and for tissue and organ development, including brain. In addition to the potent antioxidant action, it was observed that vitamin E can act as signaling and gene regulation molecule, and vitamin E incorporation into cell membranes can influence the activity of membrane-associated and integrated proteins modulating their signaling via molecular mechanisms that are independent from the antioxidant activity .TTPA in humans, and in the −/−Ttpa knock-out mouse model, are represented by neurological symptoms [Vitamin E has a pivotal role in brain. Indeed, the pathological manifestations of the familial syndrome ataxia with vitamin E deficiency (AVED), caused by mutations in the α-tocopherol transfer protein (TTP) gene symptoms . TTP regsymptoms . Moreovesymptoms .−/−Ttpa mice were used, in which the TTP protein is lacking and represent a model of vitamin E deficiency and oxidative stress. Even if these mice received a diet supplemented with α-tocopherol, only plasma levels of vitamin E appeared normal, while its levels in the cerebellum and prefrontal cortex showed only a slight increase, suggesting a main role of TTP in the brain. Vitamin E deficiency induced an increase in cerebellar oxidative stress, as suggested by the high levels of protein nitrosylation, prevented in part by dietary vitamin E supplementation. Moreover, vitamin E deficiency caused cellular atrophy and diminished dendritic branching of Purkinje neurons associated with cognitive deficits that were prevented by vitamin E supplementation. All together, these data suggested that an adequate vitamin E level is essential for anatomic integrity and physiological function of the Purkinje neurons [−/−Ttpa mouse brains showed a significant increase, while α-tocopherol supplementation prevented lipid peroxidation and neurological symptom development [The problems associated with vitamin E deficits may be caused by the loss of its antioxidant protection, and consequently ROS accumulation that compromised the integrity and function of biological membranes. At cellular level, vitamin E deficiency caused degeneration of neurons. In particular to study the importance of α-tocopherol in the cerebellum,  neurons . Further neurons ,34,35. Ielopment  demonstrelopment .1–42 induced protein oxidation, ROS production, and neurotoxicity in primary rat embryonic hippocampal neuronal culture, maybe through the scavenging of Aβ-induced free radicals [2O2, lipid peroxidation and thiobarbituric acid reactive substances (TBARS) production, and was accompanied by apoptosis, increasing caspase 3 activation, cytochrome c release and cleavage of poly-ADP ribose polymerase (PARP). Interestingly, these effects were prevented by α-tocopherol [1–42 caused a significant reduction in phospholipid and ubiquinone-10 levels, as well as in α3 and α7 subunit levels of nicotinic acetylcholine receptors (nAChRs). However, vitamin E pretreatment partially prevented these alterations. These results indicated that Aβ induced lipid peroxidation may cause the alterations in membrane lipid composition and the decreased expression of nAChRs involved in the pathogenesis of AD [Interestingly, studies in vitro showed that vitamin E may be able to counteract oxidative stress induced by Aβ ,38. In pradicals  showed tcopherol . In addiis of AD .Moreover, oxidative stress induced Aβ-like substances and apoptosis in rat hippocampus, resulting in cognitive deficit. However, memory deficits and apoptosis were prevented by vitamin E supplementation, thanks to its antioxidant action .1–42 reduced the surface expression of the main glutamate transporter in adult brain, glutamate transporter-1 (GLT-1), in mouse astrocytes through oxidative stress, and caused GLT-1 ubiquitination and mislocalization at the cell membranes, provoking a prolonged extracellular lifetime of released glutamate, but this effect was prevented by the water-soluble analog of vitamin E trolox [Aβ1–40 blocked the development and differentiation of NPCs, associated with elevated levels of protein carbonyls and lipid peroxidation products, such as HNE and malondialdehyde (MDA) and accumulated Aβ monomers and oligomers. Treatment with vitamin E partially abolished these effects, suggesting that Aβ influences neurogenesis through oxidative damage, but vitamin E was not able to counteract the reduced formation of neuronal processes [Aβ-induced oxidative damage caused neurotoxicity also against neural progenitor cells (NPCs). Indeed, Aβrocesses .However, some of the biological effects of vitamin E did not depend on its antioxidant action, but it influenced the expression of genes involved in AD. Indeed, it was found that vitamin E deficiency influenced gene expression in the hippocampus and in particular, the genes regulated by vitamin E were associated with hormones and hormone metabolism, nerve growth factor (NGF), apoptosis, dopaminergic neurotransmission, clearance of Aβ and advance glycated end products . In partThe important role of oxidative stress in AD was already demonstrated, but it is important to notice that oxidative stress occurs early in AD, even before Aβ plaque formation. Indeed, using different AD models, it was demonstrated the increase of oxidative stress, as indicated by increased lipid peroxidation ,46. More1–42, showing that its oral administration, from three days before Aβ infusion, prevented learning and memory deficits [The action of vitamin E was tested against Aβ toxicity in rats infused with Aβdeficits . However1–42-treated mice showed memory deficits and increased oxidative stress, demonstrated by the increased activities of the cytosolic Cu, Zn-SOD and mitochondrial Mn-SOD in the hippocampus and cerebral cortex in association with increases in MDA, a marker of lipid peroxidation, and protein carbonyl in the same areas. However, α-tocopherol, administrated orally, from seven days before Aβ injection, significantly attenuated Aβ1–42 induced effects, including the memory deficits [Aβdeficits .−/−TTPTtpa knockout mice, where the lack of α-tocopherol in the brain caused oxidative stress, with APPsw AD transgenic mice. The double-mutant −/−APPswTtpa mice receiving a α-tocopherol-deficient diet developed a more severe and precocious cognitive dysfunction in the Morris water maze, novel-object recognition and contextual fear conditioning tests compared to APPsw mice, together with an increase in Aβ deposits. However, α-tocopherol supplementation to −/−APPswTtpa mice partially prevented the development of cognitive dysfunction and decreased Aβ deposits in cortex and hippocampus. However, even if an improvement of the condition was obtained, the recovery was not complete, maybe because only 10% of α-tocopherol can be recovered in the brain of −/−Ttpa mice even after α-tocopherol supplementation compared with wild-type (WT) mice [−/−APPswTtpa mouse brain and plasma, that was reduced by α-tocopherol supplemented diet [APP mRNA and protein levels of C-terminal fragments of APP-β, -α, and -γ/ε in the brain of −/−APPswTtpa mice were not increased compared with APPsw mice. Instead, Aβ accumulation was due to a decreased Aβ clearance. Indeed, the expression level of the Aβ-degrading peptidase IDE was decreased in −/−Ttpa mouse brain. In addition, the Aβ aggregation increased in the brain of the −/−Ttpa mice compared with WT. −/−APPswTtpa mice showed a marked increase of both plasma Aβ1–40 and Aβ1–42 levels, that was partially reduced in mice receiving the α-tocopherol-supplemented diet. The Aβ accumulation in the plasma was also caused by impaired clearance from the blood. Indeed, decreased levels of LRP-1, the Aβ receptor that transports Aβ into the hepatocytes, were found in the plasma membrane fraction of −/−Ttpa mouse liver, causing a reduced Aβ clearance in the blood, and this may explain the increased Aβ levels in −/−APPswTtpa mouse plasma [Nishida et al.  studied WT) mice . Moreoveted diet . Howevere plasma . All togPLTP-knockout (PLTP-KO) mice showed an increase in oxidative stress and brain Aβ1–42 levels, while the expression of the synaptic function marker synaptophysin was reduced. Moreover, PLTP-KO mice presented memory deficits a week after Aβ25–35 injection. Dietary supplementation of vitamin E in PLTP-KO mice prevented Aβ25–35-induced memory deficits and oxidative stress [Plasma phospholipid transfer protein (PLTP) plays a main role in vitamin E transfer and its deficiency reduced brain vitamin E levels in mice. e stress .1–40 and Aβ1–42 levels and amyloid deposits in younger Tg2576 mice that received a diet supplemented with vitamin E from five months of age, but not in older AD mice, receiving the same diet from 14 months of age, when amyloid plaques are already deposited. However, vitamin E supplementation could counteract oxidative stress induced in both groups, reducing 8,12-iso-iPF2α-VI levels in cortex and hippocampus as a result of the increased vitamin E brain level [Sung et al.  pointed in level .Other than on Aβ deposits, vitamin E exerted a positive action on tau hyperphosphorylation, the other hallmark of AD. Indeed, transgenic mice overexpressing human tau protein developed filamentous tau aggregates in the CNS, but supplementation with α-tocopherol delayed the development of tau pathology, decreased oxidative stress and improved motor function . The proThese studies indicated that vitamin E may be able to counteract Aβ-induced effects and tau hyperphosporilation, acting in this way on both AD hallmarks. But, more important from a clinical point of view is vitamin E action on cognitive performance.APOE gene are associated with an increased risk of developing AD. Apolipoprotein E (Apo E)-deficient mice, receiving a diet supplemented with α-tocopherol for 12 months, showed a better behavioral performance compared to those receiving a normal diet. The better performance was associated with the preservation of the dendritic structure in vitamin E-treated Apo E-deficient mice. Moreover, these mice showed normal levels of lipid peroxidation and glutathione. These results indicated that the dietary supplementation with vitamin E exerted a protective action against oxidative insults in Apo E-deficient mice and prevents the deficits in cognition and neuropathologic alterations [Variants of the erations . This sterations  found therations .Moreover, the chronic treatment for 15 days with trolox showed a trend toward a reduction of oxidative stress induced from Aβ plaque and toward a reversal of the structural changes in dystrophic neurites associated with Aβ plaque . However42 fibril formation [RRR-α-tocopherol quinone, an oxidative metabolite of α-tocopherol, was reported to attenuate Aβ induced citotoxicity, inflammation and oxidative stress in vitro, blocking Aβormation . In vivoormation .Repetitive concussive brain injury (RCBI) is known to enhance brain Aβ accumulation and behavioral dysfunction, in parallel with brain lipid peroxidation . Conte eEven if α-tocopherol is the most investigated compound of the vitamin E family, more recently also tocotrienols were investigated for their protective effects, sometimes showing a more prominent action than that exerted by the most famous α-tocopherol. α-tocopherol and tocotrienol were tested against streptozotocin (STZ). In particular, intracerebroventricular injection of streptozotocin in subdiabetogenic dose in rats induced cognitive impairment and oxidative and nitrosative stress, but rats that received oral administration of α-tocopherol and tocotrienol presented an improvement of the cognitive function. Moreover, α-tocopherol and tocotrienol administration prevented the reduction of GSH and SOD and catalase activity and reduced MDA, nitrite and cholinesterase activity in the brains of STZ treated rats in a dose dependent manner. Notably, all these results showed a more potent action of tocotrienol .S-positive fibrillar type plaques in the hippocampus and cortex of APPswe/PS1dE9 double transgenic mice and improved cognitive function. However, TRF supplementation did not exert anti-inflammatory action, showing no action on the microglia activation and the authors suggested that this may be caused by insufficient dose, suboptimal method of administration, low bioavailability or late age of initiation (five months) [The tocotrienol-rich fraction (TRF), containing different vitamin E analogs obtained from palm oil was tested on APPswe/PS1dE9 double transgenic mice. The TRF was shown to be able to block Aβ fibrils and Aβ oligomers formation in vitro in a dose dependent manner. In addition, the daily TRF supplementation for 10 months mitigated Aβ depositions in the cortex and thioflavin- months) .A summary of the experimental models evaluating vitamin E supplementation is shown in In some studies, vitamin E treatment was associated with other molecules with antioxidant or anti-inflammatory effects. In this way, the different compounds can act synergistically to exert a stronger protective effect.2 (PGE2), thromboxane A2 (TxB2), and of isoprostane F2α-VI (iPF2α-VI), and protein carbonyls, as biomarkers of lipid peroxidation and protein oxidation, respectively. The anti-inflammatory and antioxidant effects were associated with the decrease of soluble and insoluble Aβ1–40 and Aβ1–42 and Aβ deposits in neocortex and hippocampus. These results showed that the two drugs had an additive effect on brain inflammation and oxidative stress [Tg2576 mice, a mouse model of AD, received a diet supplemented with α-tocopherol and indomethacin, an anti-inflammatory drug, for seven months: from 8 (before Aβ deposition) to 15 months of age (when Aβ deposits are abundant). The animals receiving the supplemented diet showed increased brain levels of vitamin E and a suppression of brain oxidative stress and inflammation, as demonstrated by the reduction of glial fibrillary acidic protein (GFAP), IL-1β, cortex and hippocampus levels of prostaglandin Ee stress .Given that there is a synergistic relationship between vitamins C and E, because vitamin C recycles vitamin E radicals regenerating its antioxidant properties , the eff1–40. The treatment with 25 mg/kg of folic acid and 250 mg/kg of α-tocopherol, or 50 mg/kg of folic acid and 500 mg/kg of α-tocopherol, induced a significant improvement of spatial learning deficits induced by Aβ1–40, while higher doses did not exert protection against the Aβ1–40-induced cognitive impairment. The combination of folic acid with α-tocopherol exerted a protective action against the Aβ1–40-induced cognitive decline through a reduction of synaptic dysfunction. Indeed, while Aβ1–40-induced synaptic loss in mice, as demonstrated by the reduction of synaptophysin levels, the treatment with folic acid and α-tocopherol prevented the reduction in synaptophysin levels in the hippocampus. Moreover, the injection of Aβ1–40 caused neuroinflammation with the activation of astrocytes and microglial cells, as indicated by the increased number of GFAP and CD68-positive cells, respectively. However, the treatment did not have effects on neuroinflammation, given that it did not influence astrocyte activation and the number of CD68-positive cells in the hippocampus. Interestingly, the combination of folic acid and α-tocopherol exerted antioxidant effects, as demonstrated by the capacity of inhibiting the Aβ1–40-induced iNOS and neuronal nitric oxide synthase (nNOS) upregulation and the prevention of the Aβ-induced neuronal death in the mouse hippocampi. Moreover, Aβ1–40 injected mice showed an increased activity of the mitochondrial complexes I, II, and IV, that was decreased by the treatment with folic acid (50 mg/kg) and α-tocopherol (500 mg/kg), except for complex II activity [The combination therapy with folic acid and α-tocopherol was evaluated in mice injected in the lateral ventricle with Aβactivity .N-acetylcysteine, α-lipoic acid and α-tocopherol to aged rats from 18 months to 22–24 months of age prevented oxidative stress, inflammation and age-dependent changes in synaptosomal parameters together with a reduction of lipid peroxidation and it improved learning and memory functions [42 in brain cortex. However, rats that received from the age of 18 months a dietary supplementation with a mix of N-acetylcysteine, α-lipoic acid and α-tocopherol showed a reduction of APP, β-secretase activity and Aβ42 compared to rats with a normal diet, while neprilysin activity increased. Moreover, the supplemented diet prevented the spatial learning and memory impairments [The administration of a combination of unctions ,71. The unctions  demonstrairments .A summary of the experimental models evaluating vitamin E supplementation together with other compounds is shown in Even if experimental studies indicated that vitamin E exerts beneficial effects in AD animal models, its efficacy in AD patients is still debated. Indeed, even if AD patients showed lower plasma and cerebrospinal fluid levels of vitamin E ,74, whilIn contrast with these data, other reports showed contrasting results, showing not convincing evidence that vitamin E treatment was able to exert positive effects in AD . A threeKryscio et al.  evaluateOne of the main concern about vitamin E treatment was that high doses may increase mortality. Indeed, a meta-analysis indicated that high doses of vitamin E (≥400 IU/day) might increase the risk of mortality . HoweverOther studies evaluated whether vitamin E from food may be able to exert beneficial effects against AD. A higher consumption of foods with an elevated content of vitamin E could modestly decrease the risk of dementia and AD  and was All these results may indicate that a combination of tocopherols and not an individual tocopherol may have a stronger neuroprotective action against AD development. For this reason, given that other tocopherols except α-tocopherol are less studied other studies are needed.Another trial did not report advantages in cognitive function in women receiving vitamin E compared to those receiving placebo. However, it is interesting to notice that vitamin E group showed less cognitive decline compared with the placebo group that included women with a low dietary intake of vitamin E (lower than 6.1 mg/day), but not compared with women receiving placebo with a high intake of dietary vitamin E .Interestingly, the combination of vitamin E with other antioxidants, such as vitamin C, was shown to be able to exert beneficial actions in AD. In particular, high intakes of vitamins E and C from food or from supplements were associated with a lower risk of cognitive decline and AD ,92,93. AGiven that previous studies did not show consistent evidence of a positive effect of vitamin E supplementation, the British Association for Psychopharmacology stated that vitamin E can not be recommended for AD treatment or prevention .Several factors may influence and explain the different and conflicting results obtained by different studies. First, vitamin E dose is an important variable that needs to be evaluated. The successful results were obtained using high doses of vitamin E. Then, in some reports, vitamin E dose used was not high enough to obtain a clinical result. However, it is not easy to determine the optimal dose and further studies are needed, but it is important to notice than high doses do not seem to be associated with increased mortality and side effects. A second important point is the vitamin E form used, given that in most cases supplements contained α-tocopherol, but it seems that other dietary tocopherols and tocotrienol, and then their combination, may be more useful to obtain a stronger neuroprotection. Another point that should be addressed is the timing of study initiation. Indeed, it is possible to speculate that an early administration may be more useful in preventing cognitive deficits, given that no study demonstrated the ability to reverse the disease process. In this context, the severity of AD is also an important factor to consider and that may influence the success of the therapy. In addition, long-term treatment seems to be necessary to obtain a significant result.Moreover, it must be considered that not all the studies considered the same parameters to evaluate the success of the vitamin E treatment, and for this reason the results of different studies cannot be compared to each other. Indeed, while some studies evaluated the capacity to carry out daily life activities, others considered the progression of MCI to AD. In addition, confounding variables cannot be excluded, such the use of other drugs or the presence of concomitant diseases, that may influence the results.Notably, an interesting hypothesis was proposed by Cervantes and Ulatowski  in theirThe number of people affected by AD is continuously increasing and, for this reason, a treatment able to prevent or delay the progression of the disease is needed. Evidence showed that oxidative stress plays a main role in AD pathology. In particular, it was observed that Aβ takes part in increasing oxidative stress, causing ROS production and leading to lipid peroxidation and protein oxidation. In turn, oxidative stress promotes Aβ production. Given this, antioxidant compounds may be helpful in the prevention/treatment of AD. Vitamin E is one of the most important antioxidant and some data indicated that it could counteract Aβ-induced oxidative stress. Evidence from preclinical studies showed that vitamin E administration may be beneficial in AD. Indeed, vitamin E is not only able to reduce Aβ-induced oxidative stress, but also able to improve memory and cognitive deficits. In particular, the precocious and prolonged administration seemed to be related to better results. Even if α-tocopherol is the most investigated member of vitamin E family, also tocotrienols showed good results in AD animal models, exerting in some cases a stronger action than α-tocopherol. Furthermore, the combination of vitamin E with other compounds with anti-inflammatory or antioxidant activity, that act synergistically, may be useful for the treatment of AD. However, clinical trials provided conflicting results about vitamin E efficacy in the prevention and/or treatment of AD. High vitamin E doses and prolonged supplementation seem to be associated with better results. Moreover, a higher intake of foods rich in vitamin E, which contain a combination of different forms of vitamin E, was associated with a better cognitive function. However, further studies are required to obtain clear results about vitamin E efficacy in AD. In this context, trials evaluating different doses and forms of vitamin E and their combination could be helpful.


The soil treated with a mixture of compost and FYM inputs had the maximum urease activity of all tillage treatments. Organically manured treatment (F4) showed more activity in dehydrogenase (85.7 μg PNP g−1 h−1), acid phosphatase (199.1 μg PNP g−1 h−1), and alkaline phosphatase (3183.6 μg PNP g−1 h−1) compared to those treated with chemical fertilizers. In NT-F4 treatment, using on-farm inputs is most suitable for sustainable management and improvement in soil biological activities in soybean cultivation. We concluded that applying organic manures and employing reduced tillage systems increased soil microbial biomass and enzyme activities.Tillage operation and fertilizer type play important roles in soil properties as far as soil microbial condition is concerned. Information regarding the simultaneous evaluation of the effect of long-term tillage and fertilization on the soil microbial traits of soybean farms is not available. Accordingly, it was hypothesized that, the microbial biomass and enzyme activity, more often than not, respond quickly to changes in soil tillage and fertilization. Therefore, the experiments were aimed at analyzing the responses of soil microbial traits to tillage and fertilization in a soybean field in Kurdistan University, Iran. The field soil is categorized into coarse Loamy, mixed, superactive, calcareous, and mesic Typic Xerorthents. The experiments were arranged in split plot, based on randomized complete block design with three replications. Main plots consisted of long-term (since 2002) tillage systems including conventional tillage (CT), minimum tillage (MT), and no-tillage (NT). Eight fertilization methods were employed in the sub-plots, including (F1): farmyard manure (FYM); (F2): compost; (F3): chemical fertilizers; (F4): FYM + compost; (F5): FYM + chemical fertilizers; (F6): compost + chemical fertilizers; (F7): FYM + compost + chemical fertilizers and (F8): Control (without fertilizer). The highest microbial biomass carbon (385.1 μg) was observed in NT-F4 treatment. The NT treatment comparatively recorded higher values of acid phosphatase (189.1 μg PNP g Tillage operations, which use moldboard plowing and disking to prepare soil for sowing, can decrease soil microbial activity and organic matter ; (F2): 2500 kg compost ha−1; (F3): 90 kg triple super phosphate ha−1 + 60 kg Urea ha−1; (F4): 2700 kg FYM ha−1 + 1250 kg compost ha−1; (F5): 2700 kg FYM ha−1 + 45 kg triple super phosphate ha−1 + 30 kg Urea ha−1; (F6): 1250 kg compost ha−1 + 45 kg triple super phosphate ha−1 + 30 kg Urea ha−1; (F7): 1800 kg FYM ha−1 + 833 kg compost ha−1 + 30 kg triple super phosphate ha−1 + 20 kg Urea ha−1 and (F8) control (without fertilizer). Organic fertilizers were also analyzed for chemical and nutrient properties according to Peters et al. (2 (full bloom) development stage. Adopting a plant spacing of 40 and 20 cm, the soybean seeds were sown on May 10 and 12 in 2012 and 2013 growing seasons employing Williams's cultivar, which is an early maturing soybean cultivar whose growth type is indeterminate, while its growth duration is about 135 days. The field was irrigated twice with a 7–9 day interval for a better growth and establishment of the seeds. It was also irrigated at stemming and flowering along with fertilization, and at podding and grain filling. Total water applied by irrigation during the crop cycle was about 500 mm. Weeds were removed manually in all treatments.The experiments related to the study were carried out at the Research Farm of Kurdistan University, Sanandaj, Iran, an area located at a latitude of 35°15′ N, a longitude of 47°1′ E and an altitude of 1300 m above the mean sea level. This site was chosen owing to its long-term tillage history. Divided into three tillage managements in 2002, this area had already been farmed prior to our experiments. Table s et al.  method. 2Cr2O7 (1.5 N) in an acid environment with H2SO4  as the substrate. Two milliliters of 0.5 M sodium acetate buffer (pH 5.5) were added to 0.5 g of soil using acetic acid and 0.5 mL of substrate and then incubated at 37°C for 90 min. The reaction was terminated by a cooling degree of 2°C for 15 min. Next, 0.5 mL of 0.5 M CaCl2, and 2 mL of 0.5 M NaOH were added, and the mixture was centrifuged at 2287 × g for 5 min. The p-nitrophenol was determined by spectrophotometry at 398 nm  was measured via spectrophotometer, employing oxidizing organic carbon with Kp < 0.05). For significant interactions, slicing was used. Prior to the combined analysis of variance, Bartlett's  vs. Other fertilized treatments .Organic manure treatments  vs. Chemical or combined fertilizers .Compost bearing treatments  vs. Treatments without compost .Farmyard manure bearing treatments  vs. Treatments without farmyard manure .None of the interactions between the experimental factors and year were statistically significant for studied variables Table . TherefoTillage and fertilization treatments and the interaction of tillage × fertilization had significant effects on MBC. SOC, however, was not affected by tillage and fertilization Table . Moreove−1 h−1) while the lowest were observed in the CT treatment (125.9 and 1989.6 μg PNP g−1 h−1)  led to higher acid and alkaline phosphatase activities compared to treatments with chemical fertilizers. The highest acid and alkaline phosphatase activities were found in F4 treatment. There was no significant difference, however, between F4 and F7 as far as alkaline phosphatase activity is concerned Table . The ort led to h−1 h−1) compared with MT (62.8 μg PNP g−1 h−1) and CT (63.7 μg PNP g−1 h−1) treatments (Table Dehydrogenase activity was significantly higher in soils receiving organic fertilizers F4) than those under chemical fertilizer treatment. However, there was no significant difference among F4 and F2 and F7 treatments as far as dehydrogenase activity is concerned Table . Dehydro than thots Table . Further−1 chemical nitrogen fertilizer and decreased employing more than 120 kg ha−1. In China, results of a certain study revealed that chemical nitrogen fertilizers of more than 160 kg ha−1 had negative effects on soil microbial biomass in grassland , linearly increased the phosphatase activities  have great potentials for stabilizing production in semiarid conditions and have higher grain yields than the CT (Moreno et al., The present study provides information on soil microbial biomass parameters as influenced by tillage, organic and inorganic fertilization in soybean production conditions. We concluded that on a short-term basis, MBC and soil enzyme activities are dependent on whether organic manures or chemical fertilizers are applied. Soil microbial biomass and enzymatic properties were also closely related with the carbon inputs. These differences were most pronounced between no-tillage and conventional and reduced tillage. In NT-F4 treatment, using on-farm inputs is most suitable for sustainable management and improvement in soil biological activities in soybean cultivation where a low input system can be carried out. Our results support the hypothesis that the microbial biomass and enzyme activity, more often than not, respond quickly to changes in soil tillage and fertilization. The NT system, which largely increases the surface of crop residues, is the most effective method for improving soil microbial biomass of carbon and enzymes activities in a relatively short term and for sustaining agricultural ecosystems.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In multicellular organisms, cell motility is central in all morphogenetic processes, tissue maintenance, wound healing and immune surveillance. Hence, the control of cell motion is a major demand in the creation of artificial tissues and organs. Here, cell migration assays on plastic 2D surfaces involving normal (MDCK) and tumoral (B16F10) epithelial cell lines were performed varying the initial density of plated cells. Through time-lapse microscopy quantities such as speed distributions, velocity autocorrelations and spatial correlations, as well as the scaling of mean-squared displacements were determined. We find that these cells exhibit anomalous diffusion with q-Weibull speed distributions that evolves non-monotonically to a Maxwellian distribution as the initial density of plated cells increases. Although short-ranged spatial velocity correlations mark the formation of small cell clusters, the emergence of collective motion was not observed. Finally, simulational results from a correlated random walk and the Vicsek model of collective dynamics evidence that fluctuations in cell velocity orientations are sufficient to produce q-Weibull speed distributions seen in our migration assays. All nucBesides their biological relevance, motile cells constitute a preeminent topic of interest in the physics of active systems , 6. The From the statistical physicist stand point, individual cell migration can be mapped on a search process for targets , which can be detected by the cell only within a limited spatial range . In the Dictyostelium cv(τ)=1N-p(α) was defined as the fraction of trajectory steps for which their instantaneous turn angle αi lies between kΔα and (k + 1)Δα relative to their previous steps. The turn angle is αi = θi − θi−1, where θi is the orientation of the displacement i, α = (αmax − αmin)/50 were built. As in reference q)1/ and their slopes γe indicated. In the insets, we have fitted the whole set of experimental data for the average mean-squared displacements by using a nonextensive statistics scenario in which a crossover between two different power-laws is assumed  is a white noise with the intensity ξ ranging in the interval . For the correlated random walk model, ϕi(t + Δt) = θi(t), i. e., the polar angle specifying the direction of motion of the particle i at the previous time. In turn, for the Vicsek model,Arg returns the polar angle specifying the direction of the average vector j, and ϕi corresponds to an orientation parallel to the mean velocity of the neighboring particles (including i) located within a radius r0 around i. So, the evolution in time of particle positions is described by the mapt ≡ 1.Concerning the origin of the q-Weibull distributions for cell speeds, we focused on a major trait of cell migration: persistence. Its effect on minimal models of single and collective cell motion was studied through computer simulations. Specifically, two models were considered, namely, a correlated random walk and the Vicsek model . In bothδ-function. We refer to it as the “microscopic” speed distribution. However, the sampling time interval in which the particle positions are recorded in an experiment is usually much larger than the microscopic time interval Δt. Indeed, the experimentally measured velocity is τ is the sampling time interval. A speed distribution p(v), termed as “macroscopic”, corresponds to such velocities.Since in both models all particles move with a constant speed, the associated speed distribution is a Dirac τ = 50 was fixed and three noise intensities ξ = 0.45π, 0.66π, and π were tested. For both models the stationary speed distributions are q-Weibull in the presence of noise (ξ ≠ 0), but a Dirac δ-function in the absence of noise (ξ = 0). In particular, at the random walk limit (ξ = π), the speed distributions converge to Weibull functions with q → 1 and r = 2, i. e., 2D Maxwellians.In Figs The motility of epithelial cell lines MDCK (non-tumorigenic) and B16F10  in the absence of external chemotactic signals (random conditions) was investigated through time-lapse microscopy. As can be seen in Figs Regardless this diversity of individual cell behavior, a well defined average motility pattern emerges: these epithelial cells migrate either subdiffusively or normally at short time scales, but performs a superdiffusion at large time scales. Furthermore, the speed distributions for both MDCK and B16F10 cells are q-Weibull and the cell velocities autocorrelations are q-exponentials with very short characteristic correlation times. Concerning the non-Gaussian migration of these cells, three main results deserve special attention.r2〉 ∼ tγ, with γ ≤ 1) at short and a superdiffusive motion  at long time scales. However, for MDCK cells plated at the lower density of 1190 cells per cm2, the mean-squared displacement exhibit, as shown in Firstly, two average regimes associated to directed motion with angular fluctuations are dominant for B16F10 cells even at the low densities tested. Single B16F10 cells migrating under external chemotactic gradients or experimenting the signalling from multiple cell clusters randomly dispersed on the culture plate , performr2〉, if for these cells the ratio between speed and angular change rates is very small. It is worth to mention that for animal cells in culture is very possible that speed and angular fluctuations are correlated, therefore altering the aforementioned sketch.These authors demonstrated that the interplay of independent speed and angular fluctuations gives rise to a sequence of alternating regimes of motion for two distinct angular dynamics correspondent to persistent and directed random walks. Moreover, they showed that such complex transients are not observed when speed fluctuations are absent. Therefore, our results are an experimental evidence of complex transients similar to those predicted by Peruani and Morelli . If onlyq and r characterizing the q-Weibull functions vary. Our experimental data and model simulations indicate that q → 1 and r → 2, leading to the speed distribution p(v) ∼ v exp(−kv2) (k a constant). This Weibull distribution is the two-dimensional version of the Maxwellian p(v) ∼ v2 exp(−kv2) (k = m/2kBT) valid not only for a three-dimensional classical ideal gas, but also for any system in which quantum effects are negligible, independently on the correlations introduced by the conservative interactions between their particles [D Maxwellian, the relevance of these non-conservative interactions decreases as the density of cells increases. Hence, migrating cells tend to perform Brownian-like motions at asymptotically large cell densities. Further support to this scenario is provided by our findings that, as the cell density rises, the characteristic lengths of directional flights decrease whereas those associated to reorientation flights increase.Secondly, the speeds of MDCK and B16F10 cells are q-Weibull distributed. Furthermore, as the density of plated cells increases, the parameters articles . In the Regarding cell speed distributions, our present findings improves and corrects our previous published results . The folD rigid substrate without external chemotactic sources or mechanical guidance, these epithelial cells can not self-organize themselves into ordered collective motions. In contrast, as observed by Angelini et al. [Additionally, MDCK and B16F10 cells do not exhibit long-range spatio-temporal velocity correlations. When randomly dispersed and crawling onto a 2i et al. , onto soi et al. , 30. TheD Maxwellian (q → 1 and r = 2) limit associated to a unbiased random walk with a turn angle fluctuation range of 2π. Alignment interactions between particles can elicit or reinforce persistent, collective motions and, therefore, generate q-Weibull speed distributions. Our simulational results for the Vicsek model confirm this. In living active matter, interactions such as steric exclusion in suspensions of bacteria [Thirdly, our simulations demonstrate that persistence is the simplest yet sufficient ingredient to generate q-Weibull distributions for cell speeds. Contrasting to our previously reported results , no corrbacteria , hydrodybacteria , 33, or bacteria  are alsoD rigid substrates and under random conditions were characterized through time-lapse microscopy. The major findings are that these cell lines exhibit anomalous diffusion with q-Weibull speed distributions and short-ranged persistent motions comprised by directional flights with contour lengths exponentially distributed. This persistence is a sufficient feature to generate q-Weibull speed distributions, as our simulations demonstrate. Also, fluctuations in cell speeds are responsible for a complex behavior of the mean-squared deviation which can exhibits alternating sub- and superdiffusive regimes at low densities of plated cells. Finally, it seems that these epithelial cells do not self-organize into collective motions when plated dispersedly on a rigid plastic substrate and in the absence of any extrinsic chemotactic gradient and mechanical cues. At the cell densities tested, which are far from confluence, spatial pair and speed correlations indicate only the formation of small clusters involving close neighbor cells. This result may have relevance for tissue engineering and the design of materials to interact with cells because the properly colonization of rigid prosthetic devices or inorganic interfaces by cells through self-organization is maybe hardly constrained.The migration of epithelial cells MDCK  and B16F10  onto 2S1 PDFIn the PDF we can check the extra results concerning: bin sizes and speed distributions, mean squared displacements for distinct migratory phenotypes and turn angle distributions.(PDF)Click here for additional data file.S1 Filecel/cm2. (available in http://www.posfisicaaplicada.ufv.br/?pageid=2714).Individual trajectories of each cell over time for density of 20 (ZIP)Click here for additional data file.S2 Filecel/cm2. (available in http://www.posfisicaaplicada.ufv.br/?pageid=2714).Individual trajectories of each cell over time for density of 100 (ZIP)Click here for additional data file.S3 Filecel/cm2. (available in http://www.posfisicaaplicada.ufv.br/?pageid=2714).Individual trajectories of each cell over time for density of 2000 (ZIP)Click here for additional data file.S4 Filecel/cm2. (available in http://www.posfisicaaplicada.ufv.br/?pageid=2714).Individual trajectories of each cell over time for density of 1000 (ZIP)Click here for additional data file.S5 Filecel/cm2. (available in http://www.posfisicaaplicada.ufv.br/?pageid=2714).Individual trajectories of each cell over time for density of 1190 (ZIP)Click here for additional data file.S6 Filecel/cm2. (available in http://www.posfisicaaplicada.ufv.br/?pageid=2714).Individual trajectories of each cell over time for density of 11900 (ZIP)Click here for additional data file.S1 Videohttp://www.posfisicaaplicada.ufv.br/?pageid=2714).This film was obtained with superposition of all the pictures in all the time. (available in (WMV)Click here for additional data file.S2 Videohttp://www.posfisicaaplicada.ufv.br/?pageid=2714).This film was obtained used the position of particles in a simulation whith Vicsek Model and r = 2.80rad in all time. (available in (WMV)Click here for additional data file.


The ICD-10 categories of the diagnosis “perinatal asphyxia” are defined by clinical signs and a 1-minute Apgar score value. However, the modern conception is more complex and considers metabolic values related to the clinical state. A lack of consistency between the former clinical and the latter encoded diagnosis poses questions over the validity of the data. Our aim was to establish a refined classification which is able to distinctly separate cases according to clinical criteria and financial resource consumption. The hypothesis of the study is that outdated ICD-10 definitions result in differences between the encoded diagnosis asphyxia and the medical diagnosis referring to the clinical context.Routinely collected health data  of the University Hospital of Bern were used. The study population was chosen by selected ICD codes, the encoded and the clinical diagnosis were analyzed and each case was reevaluated. The new method categorizes the diagnoses of perinatal asphyxia into the following groups: mild, moderate and severe asphyxia, metabolic acidosis and normal clinical findings. The differences of total costs per case were determined by using one-way analysis of variance.The study population included 622 cases . By applying the new method, the diagnosis asphyxia could be ruled out with a high probability in 47% of cases and the variance of case related costs  = 55.84, p < 0.001, multiple R-squared = 0.312, p < 0.001) could be best explained. The classification of the severity of asphyxia could clearly be linked to the complexity of cases.The refined coding method provides clearly defined diagnoses groups and has the strongest effect on the distribution of costs. It improves the diagnosis accuracy of perinatal asphyxia concerning clinical practice, research and reimbursement. Routinely collected health data (encoded data) are being increasingly used for research purposes. Hospitals in Switzerland are obliged to submit encoded data to the Federal Office of Statistics on an annual basis, enabling publication of epidemiological and economic health care statistics. Both kinds of statistics are influenced by the content and quality of the data. However, encoded data may not be accurate for describing the clinical picture of diseases . This isTo overcome this problem, Switzerland, as well as other countries , elaborated individual coding guidelines, which serve national purposes. The Swiss guidelines are published annually by the Federal Office of Statistics . As ICD The ICD-10 WHO definition of „birth asphyxia”as „failing to initiate and sustain breathing at birth” is speciThe codes of category P21 “asphyxia” are defined in the ICD-10 by the 1-minute Apgar score and additionally by some of the individual elements of the 1-minute Apgar score, which are meant to reflect the severity of mild, moderate and severe asphyxia .Additionally, the exclusion term “Excl.: intrauterine hypoxia or asphyxia (P20.-)” affects the subcategory of the P21 codes, providing rules for the interrelation of codes .The clinical definition of the term “birth asphyxia” on the other hand has changed over the past 20 years from „failing to initiate and sustain breathing at birth”to „intrapartum-related hypoxia“. Both definitions are complex and open to interpretation . Anne CCSevere asphyxia is associated with multiple organ failure including hypoxic encephalopathy . The AmeThe different definitions  not only influence the quality and comparability of statistical reports but also have a high impact on reimbursement of hospital services in inpatient care.Since 2012, the federal law of reimbursement for acute inpatient care SwissDRG (Swiss Diagnosis Related Groups) has been based on payment rate-setting mechanisms using standardized cost data and classification into diagnosis-related groups .Mandated by federal law the CMO (Case Mix Office) SwissDRG calculates annual “case-based” or “case-mix-based”rates of the DRG price and reimbursement using both encoded data  and patient-level costs through regression models. This allows classification of patients into clinically meaningful groups which consume similar health-care resources. Different diagnoses are likely to result in significantly different resource consumption  during an inpatient episode. DRGs have differing levels of resource consumption and are split on the basis of CCL or on the basis of certain functions , thus, presenting a certain relative weight (= severity weight).The inconsistencies between the definitions of the clinical diagnosis and ICD code cause an inadequate assignment of costs and resources, especially with regard to the severity of disease.In acknowledgment of inconsistencies in the correct allocation of resources corresponding to the severity of the diagnosis, the Federal Office of Statistics assimilated the coding guidelines upon the request of the authors/University Hospital of Bern. The application was put forward in 2014, revised by advisory boards and implemented in 2016 , . S1 Fig.The refined guidelines were based on standard values from literature, e.g. a low 5-minute Apgar score as a high-risk marker in association with severe fetal acidemia or intubation within the first hour of life as highest risk for developing seizures secondary to perinatal asphyxia , 27. By Our aim was to specify the ICD definition of perinatal asphyxia and to develop a refined method of an accurate encoding of the diagnosis perinatal asphyxia Thus the refined coding method improved the accuracy of diagnosis of perinatal asphyxia concerning clinical practice, research and reimbursement.The University Hospital of Bern-Inselspital is a tertiary care center, specializing in gynecology and obstetrics, pediatrics incl. PICU (pediatric intensive care unit), neonatology incl. NICU  and NIMC  and pediatric surgery providing both in- and outpatient care.® [Shortly after discharge from hospital the inpatient case is encoded by medical coding specialists based on the information received from the electronic medical record. The data  has to be submitted for the reimbursement process (SwissDRG) and in-house and national statistics (Medizinische Statistik der Krankenhäuser) whereby it passes several quality checks. Case related costs are recorded according to REKOLE® , the staThe International Statistical Classification of Diseases and Related Health Problems ICD-10 German Modification (GM)  codes weThe encoded medical data and the data of case related costs are linked in a QlikView database, and clinical data (excel database) are manually linked through case identification number. In order to assess incidence, this data is compared to the national statistics provided by the Federal Office of Statistics.To derive information on the course of treatment administered per patient, all medical records were analyzed (first and second author). The information on treatment procedures was encoded according to the CHOP (28). We classified treatment into three groups based on available information: 1) CHOP codes which indicated mechanical ventilation; 2) CHOP codes which indicated a systemic hypothermia; and 3) CHOP codes which indicated significant OR (operation room) procedures .The quality of encoded data was validated by evaluating the accuracy of the determined codes with the coding guidelines of admission year (first author). The values of biochemical analyses, neurological scores and clinical diagnosis were extracted (second author) using case ID (case identification number) as a unique identifier. The datasets were anonymized.The medical statistic dataset included information on encoded diagnosis, procedures, gestational age, birth weight, age at admission (outborn), weight at admission (outborn), length of stay and DRG.The clinical information system included variables such as medical diagnosis, Apgar score at 1, 5 and 10 minutes of age, Sarnat stage, HIE therapy, biochemical values in the first hour postpartum, lowest values of pH , BE , lactate . The values of inborn newborns were extracted from electronic laboratory records, the values of outborn patients from admission letters. Unavailable data were labeled as ‘missing value’.Automated coding was created through excel macro demonstrating cut- off values of Apgar score, pH, BE, lactate und Sarnat stage per model with regard to the previously defined criteria per case. The corresponding diagnosis was encoded once a minimum of criteria was fulfilled. The relevant variables and values were documented. The excel macro was manually checked at random sampling, comparing the results to those of the automated excel macro, being identical in 100% of the cases. The cases with HIE and hypothermia served as control group.The following models were both defined based on previously existing coding guidelines (effective from 2012–2016): „Original Coding”= originally encoded by the catalogues being used at that time and coding guidelines , and „KHB.2016”= encoded by catalogues and Swiss Coding Guideline (KHB) used in 2016.Criteria of model “KHB.2016” were verified by identifying both advantages and disadvantages with respect to a consistent classification of all cases. By encoding according to the results of this analysis, the criteria of the newly defined model (“Model Matrix”) were adapted, The most important RCTs concerning hypothermia treatment and national and international criteria of indication were reviewed and taken into consideration when defining „Model Matrix“. By refining the Coding Guidelines of 2016, the group classifications of mild, moderate and severe asphyxia diagnosis in P21, as well as the identification of further clinical states to be included in the P20 encoding were made possible. Certain definitions were assigned to code P20 in order to include the encoding of metabolic values (acidosis) lacking neurological complications. The newly classified diagnosis groups were analyzed  in respect to each model, corresponding to an overview of criteria for hypothermia.Certain criteria of model “KHB.2016”, such as „necessity of intensive care treatment in neonatal intensive care unit”or „ventilation”which did not adequately correspond to a diagnosis, but rather reflected an issue of infrastructure or a behavioral intention, were not considered for the revision.As there is no clearly established distinction between mild and moderate asphyxia, the low cut-off value was based on a review of patient records and audit case study and a mean ±SD pH of all newborn between pH 7.15 and 7.20 was calculated defining the low cut-off value at pH of 7.15 .As a result of the analysis the „Model Matrix”was developed.Diagnoses of category P21.- birth asphyxia can be encoded when fulfilling the following criteria :At least 3 of the criteria mentioned below must be fulfilled:5-minute Apgar score ≤ 5severe acidosis during first hour of life: pH ≤ 7.00 basedeficit ≤ -16 mmol/L in UV or UA or during first hour of lifemoderate to severe encephalopathy (Sarnat stage II—III)lactate ≥12 mmol/L in UV or UA or during first hour of lifeAt least 2 of the criteria mentioned below must be fulfilled:5- minute Apgar score ≤ 7moderate acidosis during first hour of life: pH < 7.15 mild to moderate encephalopathy (Sarnat stage I—II)Both of the two criteria mentioned below must be fulfilled:5- minute Apgar score ≤ 7 andlowest value 1 hour of life pH ≥ 7.15 Metabolic acidosis without clinical impairment (i.e. asphyxia)5- minute Apgar score > 7moderate acidosis during first hour of life: pH < 7.15 5- minute Apgar score > 7Lowest value 1 hour of life pH ≥ 7.15 The originally encoded diagnoses were adjusted manually in the dataset of medical statistic MS  accordinThe datasets were regrouped using batch grouping , revenuewww.r-project.org, packages ggplot2, car, pastecs, Rcmdr), Notepad++, v. 6.7.5, free software.Medical Coding Software SAP IS-H Klinischer Arbeitsplatz, Medical Coding Tool ID Diacos, Clinical Data Phoenix CGM, Business Data WareHouse SAP BW, Microsoft Excel 2010, R (The Ethics Committee of the Canton Bern approved our study (KEK-Nr. Req-2016-00025). Informed consent was not necessary, as the analyses were done with routine clinical and financial data from our hospital for quality assurance purposes.The number of diagnoses P20, P21 and P91 in Switzerland as well as the number of births from 2004–2014 were derived from the national statistic (Medizinische Statistik der Krankenhäuser) and incidence was calculated  Tables.The resulting incidence for diagnoses P20.-, P21.- and P91.6 is shown in Of the 622 cases 452 were inborn, and 170 were outborn. The mean length of stay was 15.31 days , mean gestational age 37 5/7 weeks , mean birth weight 2870 g . HIE was diagnosed in 98 newborns, Sarnat Stage documented as I, II, III in 31, 44, 23 newborns respectively. 63 newborns were treated with hypothermia, of which 55 were outborn . 227 patients received mechanical ventilation or CPAP (continuous positive airway pressure), 25 patients underwent significant OR procedures.Clinical and biochemical findings such as Apgar score are shown in S1 Data® [The case related total costs were calculated by the national cost accounting method REKOLE® . Income ® , the proThe classification of the cases into distinct DRGs changed by revision of the encoded diagnoses, see flow chart The originally encoded diagnosis P20.0 showed a heterogeneous distribution to all groups from severe asphyxia to normal clinical finding . Most ofThe diagnosis P21.0 „severe asphyxia”encoded according to “KHB.2016” and “Model Matrix” showed the same results. The cases were identical, except for one case  which was allocated in group P21.1 “Model Matrix” because of the 10 min Apgar score . 43 of patients in group P21.0 „KHB.2016”and „Model Matrix”received systemic hypothermia treatment .30 cases of group P21.9 encoded according to „KHB.2016”could be mapped to group P21.1 “Model Matrix” (including 7 with treatment of hypothermia).The reason for this relevant reallocation is the threshold value of the Apgar score and consideration of the Sarnat stage. 10 cases showing an Apgar score < 4, but pH < 7.15 were allocated in group P21.9 “KHB.2016”, as the Apgar score was “too low”. Another 11 cases with Apgar score > 3, UA pH < 7.15 and Sarnat stage I-II were mapped to P21.1 “Model Matrix”. The crucial factor was the significance of the Sarnat stage, which has no criterion in model „KHB.2016”regarding diagnosis P21.1, resulting in an allocation of cases with moderate asphyxia into group P21.9.By refining the model „KHB.2016”more specific diagnoses were applied to 30 cases of group P21.9 „birth asphyxia unspecified”(7 of them received treatment of systemic hypothermia), i.e. P21.1 „moderate asphyxia”according to model „Model Matrix“. By setting a low cut-off value for the Apgar score minimum of 4 for the diagnosis P21.1 according to “KHB.2016”, patients with low values (e.g. Apgar 0 and pH 7.15) were excluded and classified into the diagnosis group P21.9.Group P21.9 shows only 88 cases when encoded according to „Model Matrix”(418 “KHB.2016”), 54% of them preterm (48/88), which corresponds to 26% of all preterm patients at a GA < 37 WGA . 34% of cases P21.9 „Model Matrix”were born at a low birth weight < 2000 g (30/88), of these 10 < 1000 g .This group consists of 7 high complexity cases, 3 of them unattended home births / non-clinical setting with no biochemical values available 1h pp, 2 of these cases were preterm births born at a low birth weight of 1815g and 700g and 1 case was a term newborn; 3 cases were transferred from other hospitals ; 1 patient with suspected infection and transfer for surgical delivery ; 3 of 7 cases were born at term, 2 of them received hypothermia treatment, 4 did not as they were preterm.Most cases  were originally encoded in group P21.1 (142 of 318). 28 cases  were allocated to “normal clinical finding” (1- minute Apgar score 4–7), resulting in 14% of group “normal clinical finding”.Analysis of the Apgar, pH, BE calc. values was done for each diagnosis group and model  s. Regarding the diagnosis of severe asphyxia, the values proved identical  in respect to model “KHB.2016” and to “Model Matrix” and matched those of the international criteria for systemic hypothermia.We set up plot graphs for each diagnosis group and each model to get a visual impression of the distribution of cases. In the distribution graphs the Y-axis and the X-axis represent in different combinations the values of 5- minute Apgar score, UA pH and BE per model, each graph showing the cases for all diagnoses.Plot “Original Coding” : Most caPlot “KHB.2016” : The casPlot “Model Matrix” : A clustTo interpret these groups and cases, a matrix was developed which consisted of a set of two-dimensional diagrams pointing out the 3 values: 1) 5- minute Apgar score, 2) UA pH and 3) BE –S4 Figs.The diagram shows a clear distinction between values and categories and characterizes the diagnosis groups distinctly.The Income SwissDRG version 1.0–5.0 and the profit per SwissDRG 1.0–5.0 version were calculated for each model . To ensure the versions’ comparableness a fixed base rate of 11’000 CHF was set. Common logarithms were used to transform continuous variables to stabilize variance. Data were evaluated for F-distribution, and by visual inspection for skewness and kurtosis (histogram and QQ-diagram). The Shapiro-Wilk normality test was applied. Data points were evaluated for leverage by inspection of residuals vs. fitted plots using Cook’s D statistic. There was no normal distribution, the data were skewed. The large sample size of 622 cases permitted to use the strength of the central limit theorem of probability theory.Profitability was evaluated using the IQR-Method , IQR beihigh deficit case:Financial results for each case were calculated and separated into three categories: „high deficit“, „high profit”and „normal”. Cases as originally encoded and simulated according to SwissDRG version 4.0 show 46 „high profit”cases, with a total profit of 1‘427‘157.86 CHF, a mean profit per case of 31‘025 CHF (SD 16‘600 CHF). A „high deficit”was found in 64 cases, total deficit of all cases -3‘081‘090.61 CHF, mean deficit per case of -48‘142 CHF (SD 38‘936.6 CHF). The financial result of „high profit”versus „high deficit”shows a total deficit of -1‘653‘932.75 CHF. The total deficit of the billing period, of version 4.0 SwissDRG and of version 5.0 SwissDRG is -1‘380‘844.9, -2’942’591.9 and -916’838.5 CHF respectively .Total costs und log10 Costs per diagnosis and model  were calculated .Model “Original Coding”: lowest costs can be observed in the group of P20.1: mean 3.811 (SD 0.5397), similar results with the other groups: mean 4.05–4.55 (SD 0.5–0.63). Apart from group P21.0 a strong divergence of costs cannot be detected. Model „KHB.2016“: all groups show a clear tendency and distinction: P21.0, P21.1, P21.9 present declining costs. Model „Model Matrix“: Log10 costs are highest in group “not assigned”: mean 4.905 (SD 0.6764), followed by “severe asphyxia”: mean 4.564 (SD 0.414) The lowest log 10 costs appear in the group of „normal clinical finding” (mean 3.715 (SD 0.516)) and “metabolic acidosis without neurological impairment” (mean 3.752 (SD 0.475)).The Levene-Test, the Kruskal-Wallis test and the results of the one-way independent ANOVA by comparing models “KHB.2016”, “Model Matrix” and “Original Coding” show the following results, see also Boxplot log10 costs per diagnosis group “Original Coding” The effect of “Original Coding” encoding on the log10 Costs Variance: F = 40.4, p<0.0001, multiple R-squared = 0.247, adjusted R-squared = 0.2408, Levene-Test F = 1.0039, p>0.1; Kruskal-Wallis chi-squared (5) = 155.11, p-value < 0.001.The effect of “KHB.2016” encoding on the Log10 Costs Variance: F = 50, p<0.0001, multiple R-squared = 0.1391, adjusted R-squared = 0.1363, Levene-Test F = 13.483, p<0.001; Kruskal-Wallis chi-squared (2) = 93.76, p < 0.001.There was a highly significant effect of encoding according to “Model Matrix” on the log10 Costs Variance, F = 55.84, p<0.0001, multiple R-squared = 0.312, adjusted R-squared = 0.3063, Levene-Test F = 3.1798, p<0.01; Kruskal-Wallis chi-squared(5) = 197.24, p <0.001.The distribution of costs is significantly affected by the model of encoding and its corresponding criteria. The explained variation with “Original Coding”: multiple R-squared = 0.247, adjusted R-squared = 0.2408; with “KHB.2016”: multiple R-squared = 0.1391, adjusted R-squared = 0.1363; with “Model Matrix”: multiple R-squared = 0.312, adjusted R-squared = 0.3063.Due to the highest R-squared value “Model Matrix” gives the best explanation for cost variance.Through the application of the “Model Matrix” the originally encoded diagnoses of “birth asphyxia” in 622 inpatient cases treated at the Inselspital University Hospital of Bern in between 2012–2015 were evaluated. The analysis of the clinical data presents discrepancies between the medically determined diagnosis and the ICD-10 coding which could be both identified and quantified. The hypothesis that the use of outdated ICD-10 definitions resulting in differences between the encoded diagnosis asphyxia and the medical diagnosis referring to the clinical context was confirmed.Clinical signs such as „abnormal fetal heart rate“, „distress”, „meconium in liquor“, „passage of meconium” or “acidosis” are equated with ICD-10 diagnosis P20 “Intrauterine hypoxia”. Citing McLennan: „Signs of fetal compromise such as changes in fetal heart rate and passage of meconium are neither sensitive nor specific to any particular cause and only sometimes indicate damaging intrapartum hypoxia” . The ACOOver the past 20 years the US National Institute of Child Health and Human Development has been elaborating terminology and interpretation of abnormal fetal heart rate . HoweverP20.1 “intrauterine hypoxia first noted during labour and delivery” was originally the most commonly coded diagnosis . Relating to asphyxia signs and symptoms like meconium in liquor or abnormal pattern CTG had been documented in the patients’ records. However, these signs and symptoms are in general not specific enough to identify the diagnosis severe asphyxia, as they are only associated to 7.8% of cases . Most ofMetabolic acidosis determined solely from samples of umbilical artery at birth is a poor predictor of perinatal brain damage  and whenPrepartal signs such as abnormal fetal heart rate are, if not correlated with other findings, indicators but are neither medically relevant for the diagnosis of asphyxia nor for assessing the patients’ outcome postpartum.The group of cases originally encoded with diagnosis P20.0 „intrauterine hypoxia before onset of labour”(22 cases) show a high variability after recoding with regard to severity ranging from diagnosis “severe asphyxia” (27% / 6 cases) to “normal clinical finding” (36% / 8 cases).Relating facts such as a silent pattern CTG with severe asphyxia were documented in the patients‘ records, but never the term or diagnosis „intrauterine hypoxia”itself. Possible interpretations are that due to the instruction of the existing exclusion term (exclusion of P21) P20 “intrauterine hypoxia” had been encoded instead of P21 “birth asphyxia”. The ACOG, the AAP, the Task Force on Neonatal Encephalopathy and Cerebral Palsy recommend against the use of the term “birth asphyxia” unless there is clear evidence of intrapartum-related causation, as they outlined criteria which together suggest an intrapartum timing, but individually are nonspecific to asphyxia insults , 36. RefAs stated by the AAP in the “Use and Abuse of the Apgar Score” : “A low The 1-minute Apgar alone, listed as a defining but redundant element in the ICD diagnosis P21, should not be used as evidence of hypoxia causing neurological damage . AccordiOur results point out, that encoding the specific diagnosis by referring to a 1- minute Apgar score of 0–3 shows a higher correlation with the medically identified diagnosis “severe asphyxia” (28%) than the 1-minute Apgar score of 4–7 (4.2%) or other elements in the ICD like the mentioned signs “meconium in liquor”, “abnormal fetal heart rate”, “distress”. However, the validity of the 1-minute Apgar score remains uncertain.The causal relation was described by Sarnat & Sarnat in “Neonatal Encephalopathy Following Fetal Distress” : “The seBy the development of a new classification model (“Model Matrix”) a realistic cut-off point for defining pathological fetal acidemia which correlates with an increasing risk of neurological deficit was determined. This is defined as a pH of less than 7.00 and additionally a base deficit of more than 16 mmol/l , 51. A 5The most important characteristic of model “Model Matrix” is the possibility of classifying each individual patient based on the clinical and laboratory values and criteria into a distinct diagnosis group, regardless of level of care received or intended treatment. All these observations point out, that according to „Model Matrix”all criteria can be matched distinctly to one category. Considering the severity of illness the distribution of values seems coherent from visual perspective.The criteria of the diagnosis „severe asphyxia”P21.0 and of the indication of hypothermia treatment overlap, showing the following results : 5- minute Apgar 3.25, 1.92; UA pH 6.93, 0.16, UA BE -14.7, 5.16 corresponding to those of available RCT –53, Tabl80 cases were classified as severe asphyxia, 43 newborns were treated with hypothermia .Definition of criteria by “Model Matrix” includes 5-min Apgar score values ranging from 0 to 7 at a pH < 7.15 and at a Sarnat stage of I to II. Out of 154 newborns 15 were treated with hypothermia .Including the 5- minute and 10- minute Apgar score in model “KHB.2016” appears to be advantageous and should also be considered for model “Model Matrix”, especially as the Bernese hypothermia protocol is based on the 10- minute Apgar.As there is no clearly established distinction or definition of mild versus moderate asphyxia, we defined the low cut-off value, based on review of patient records and audit case study and calculated the mean ±SD pH of all newborn between pH 7.15 and 7.20. Thus, we defined the low cut-off value at pH of 7.15 .It seems necessary to distinguish clearly between the diagnosis mild asphyxia and normal clinical finding. Cases with an abnormal adaptation (5- minute Apgar <7) should be allocated in group P21.9 “mild asphyxia” according to “Model Matrix”. 52% of newborns in diagnosis group P21.9 were preterm, GA < 37 WGA (46 /88), 10 of these had a birth weight < 1000 g (11% of P21.9). Due to prematurity only 3 patients of this group received systemic hypothermia treatment. The Apgar score is less reliable in premature infants, as it directly correlates with gestational age . In premMost of the patients in this group show no apparent signs of illness. Most cases  were originally encoded in group P20.1 (142 of 318), which is due to the specified criteria such as abnormal fetal heart rate or meconium in liquor. The criteria and values set up for defining normal clinical finding in model “Model Matrix” enables coding to be more precise with respect to a distinction between normal and asphyxia. As a result of revising the cases with diagnosis P20 intrauterine hypoxia and P21 birth asphyxia according to model „Model Matrix”most cases were allocated into new groups: “normal clinical finding” (33%) and “metabolic acidosis without neurological impairment”(14%). With these two groups of patients  a clinical impact of birth hypoxia could be ruled out with a high probability. This statement could be confirmed by analyzing the patients’ records.A clear correlation between the complexity level of diagnosis and resource consumption was detected.As could be expected in regard to resource consumption, the lowest costs were observed in the group P20.1 “Original coding”. When recoded according to „Model Matrix”the cases in group “not assigned” contribute to the highest costs followed by the cases of group P21.0 “severe asphyxia”. The lowest costs can be observed in the group of „normal clinical finding” and “metabolic acidosis without neurological impairment”.Due to the highest R-squared value  “Model Matrix” gives the best explanation for cost variance of the very heterogeneous patient population which includes neonates with a gestational age of 23–44 weeks and also congenital heart defects. Other elements such as gestational age, birth weight, ventilation, systemic hypothermia and significant OR procedures were not used for revision of cases. Although these elements are important in allocating resource consumption and costs, they were not taken into consideration in this study. The aim was to outline the relevance of diagnosis birth asphyxia in respect to reimbursement under the current DRG system.In summary the analyses of “high deficit”and „high profit”cases point out that the 59 “high deficit” cases were responsible for 35% of all costs (7‘174‘875 CHF of 20‘612‘100 CHF). The resource consumption of certain diagnoses are not counterbalanced in the system of SwissDRG 5.0.85.5% of cases from category „high deficit”(n = 63) belong to group P21: P21.0 , P21.1 , P21.9 .In version SwissDRG 5.0 (2016) the codes asphyxia P21, and severe asphyxia P21.0, are not included in any of the above mentioned mechanisms of cost allocation and consistent reimbursement, only code P20.0 “intrauterine hypoxia” is paradoxically listed as function “severe problem”. The outlined inadequacy can be explained by numerous encoding of asphyxia until 2015. We do expect a better impact of the diagnoses on the explanation of variance when encoded by validated criteria. We should now concentrate on refining the distinction of diagnoses by focusing on category P21 instead of P20. Failing to demarcate codes and diagnoses clearly and in relation to resource consumption, these interdependencies lead to an inadequate assignment of costs and resources. Our study intended to enhance development of a more sufficient DRG and comprehensive reimbursement system.According to Lancet Neonatal Survival Steering Team, asphyxia, one of the major direct causes of neonatal deaths globally  , is yet National statistics were used both by The Lancet Ending Preventable Stillbirths study group, The Lancet Stillbirths in High-Income Countries Investigator Group –65 to coFrom 2004–2014 according to figures of the Medical Statistic  an incidence of 40–80 cases of asphyxia per 1000 births was recorded in Switzerland, In 2005 a national cooling register for cases with hypothermia was introduced in Switzerland , 74–77. Routinely collected health data are being increasingly used for research. Quality recommendations and standards for reporting of observational routinely-collected health data help improve the accuracy of results  , 79.In epidemiological research, ICD codes are being used as selection criteria. Considering in general the insufficient definitions and specifications of the ICD diagnoses, the discrepancy of incidence might be explained partly not only by health care quality but also by national coding.Limitations of the study included the fact that secondary data not collected as part of our study were used. With the exception of 7 cases data of all the selected cases were complete. The methodology of data collection remained constant from 2012–2015.Misclassification bias: No intention of upcoding, opportunistic coding and maximizing reimbursement can be observed as the diagnoses referring to asphyxia are irrelevant for DRG classification. Furthermore, encoded cases are revised annually and systematic in-house quality checks are performed.It has to be taken into account, that any change in definitions concerning code P20.1 may cause adjustments in practice of encoding, statistics and reimbursement. We acknowledge that the discrepancy of encoded diagnosis and medically determined diagnosis is increased by the exclusion term and by the existing definition in code P20 and should be revised.Missing variables: Information on laboratory findings of outborn patients should be obtained, the process has to be improved. But most important, the relevant biochemical values in complex cases were all registered.Our study has several strengths. We analyzed standardized data of all inpatients of our hospital, our results providing indices for university hospitals in general, to our knowledge, a unique approach in Swiss research. As the criteria can be verified, the refined model “Model Matrix” offers the advantage of being able to calculate the PPV , NPV , TP (true positive), TN (true negative) (79) with respect to diagnosis.There had been extensive encoding efforts of asphyxia from 2012 until 2015. To achieve a reasonable progress concerning the SwissDRG system, quality of data must be improved. This requires an accurate diagnosis as well as corresponding coding guidelines.Through the definition of five diagnosis groups, a distinct allocation of cases can be achieved. The newly introduced model “Model Matrix”  explains approximately 30% of cost variance of a very heterogeneous group of patients and appears highly suitable for clinical use, research and reimbursement.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.