Waxes, phospholipids, free fatty acids, peroxides, aldehydes, soap, trace metals and moisture present in crude sunflower oil have a negative effect on the oil quality and are, therefore, removed during the refining process. Waxes crystallizing at low temperatures are removed during winterization by cooling and filtration. Waxes have poor filtration characteristics and an industrial filtration process must be enhanced by the use of filtration aids, which improve filter cake structure and properties, and consequently prolong the filtration cycle. Today, traditional filtration aids  being used in the industry are frequently replaced by cellulose-based aids. The aim of this study is to examine the effect of oil filtration assisted by two cellulose-based filtration aids on the chemical parameters , oil transparency, carotenoids, and Fe and Cu content of sunflower oil obtained in an industrial horizontal pressure leaf filter. In order to investigate the mentioned parameters, the following techniques were used: gravimetric (waxes and moisture content), spectrophotometric (phospholipids and carotenoid content and oil transparency), volumetric (soaps and free fatty acids content) as well as inductively coupled plasma mass spectrometry (ICP-MS) for Fe and Cu content. An artificial neural network model (ANN) was employed for the prediction of removal efficiency based on the chemical quality, oil transparency, Fe and Cu content in oils before filtration, as well as filtration aid quantity and filtration time. Cellulose-based filtration aids had multiple beneficial effects; on average, 99.20% of waxes, 74.88% of phospholipids, 100% of soap, 7.99% of carotenoids, 16.39% of Fe and 18.33% of Cu were removed. Crude sunflower oil contains undesirable components such as phospholipids, free fatty acids, peroxides, aldehydes, trace metals, polymers, waxes, mono- and diacylglycerols, moisture and other volatile compounds . These cThe most abundant phospholipids in sunflower oil are phosphatidic acid 69%), phosphatidyl choline 13%), phosphatidyl ethanolamine (13%) and phosphatidyl inositol (5%) . The pre9%, phosp%, phosphCarotenoids are the main pigments in sunflower oil ,21. TotaFiltration of the sunflower oil during the winterization phase is aggravated by the presence of the residual substances. During the filtration, solid particles are separated from fluids, remaining on the filtration medium . Usually−2 deposited on the filter medium prior to beginning feed to the filter, is commonly used to protect the filter medium from fouling by trapping solids before they reach the medium and to provide a finer matrix to trap fine solids and assure filtrate clarity as well [Today, filtration aids consisting mainly of natural cellulose fibers are increasingly used . The adv as well . Cellulo as well ,34,35.During the winterization process, between the wax crystallization and the filtration phases, sunflower oil contains other components affecting the quality, color and other oil properties. This paper presents the results of monitoring the operation of an industrial pressure leaf filter, which is a part of the winterization process during the refining of sunflower oil. The filtration process is assisted by the addition of two filtration aids, one for pre-coat and the other for the body-feed application. The focus of the work is on the quality of the filtered oil, depending on the quality of the oil entering the filter and the amount of the added filtration aids. Waxes, moisture, phospholipids, soaps, fatty acids, carotenoids, iron (Fe) and copper (Cu) contents, as well as transparency (at wavelength of 455 nm) of the oil samples were determined. Furthermore, the main objective of this investigation was to explore the potential of forecasting the removal efficiency of mentioned parameters based on data of initial values of chemical parameters, Fe and Cu content, and also the data regarding filtration time and the quantity of filtration aid. The importance of this research is in the introduction of cellulose-based filtration aids with certain technological and ecological advantages compared to classic aids used in the oil refining process. On the other hand, since their price is higher compared to classic filtration aids, it is necessary to prove the effectiveness of these filtration aids so companies would be ready to invest. In this regard, the main objective of this work is to prove the effectiveness of cellulose-based filtration aids and to optimize the process.Helianthus annuus L.) grown in the territory of Vojvodina (north of the Republic of Serbia) in 2019.Analyzed samples were taken during the industrial refining of sunflower oil, before and after filtration, as a part of the winterization phase. Industrial processing was carried out during 2019–2020 and the industrial refining capacity was 200 tons of crude sunflower oil per day every 24 h. The obtained oil is produced from sunflower seeds    at 16 °COne commercial cellulose-based aid (FA-P) was used for the application on filters , >100 μm (5–50%), >32 μm (min 45%)), while the other commercial cellulose-based aid (FA) , >>100 μm (max 70%), >>32 μm (max 98%)) was used for the dosing during filtration .The oils were sampled before and after horizontal pressure leaf filter. A total of 22 oil filtration cycles (labeled from F_01 to F_22) were monitored. Oils sampled before horizontal pressure leaf filter had different wax content, total carotenoids content, iron and copper content. Moisture content, total phospholipids content, soap and free fatty acids content of examined oil samples before filtration also differed and depended on the efficiency of the previous refining phases . Moreover, 22 oils were sampled after the horizontal pressure leaf filters. About 1 L of oil was sampled in 1 L PET bottles, sealed with original two-part caps and stored in a refrigerator at 4 ± 2 °C before analysis.Wax content, phospholipid content, soap content, free fatty acid content, moisture content, carotenoid content, iron (Fe), copper (Cu) content and oil transparency were determined in all sunflower oil samples .n-hexane at 1–3 °C for 8 h. All the accompanying components were extracted by solvent at mentioned temperature (1–3 °C), so pure wax crystals remained on the filter paper. Later, waxes were extracted by “warm” alcohol for 4–6 h and collected in a laboratory flask. Obtained flask content was cooled to allow the waxes to crystallize and filtered. The filter paper, together with the waxes, was dried at 103 ± 2 °C and measured. Based on the obtained wax mass and initial sample mass, the wax mass fraction was calculated.The wax content in the oil samples before the horizontal pressure leaf filter was determined by gravimetric method according to Oštrić-Matijašević and Turkulov, 1973 . The keyThe wax content in the oil samples after the horizontal pressure leaf filter was too low, below the limit of quantification by the gravimetric method. In this case, the wax content was practically determined as the threshold of oil turbidity, the smallest amount of wax that causes turbidity in the oil, under the test conditions. The oil was kept for a certain time at a defined low temperature: 0, 5, 7, 12 or 15 °C, observed visually and the time required for oil turbidity to appear was recorded. This method is also used to assess the “resistance” of oil to crystallization and is most often used to control the winterization process ,37,38. TThe moisture content was measured according to ISO 662, 2016 .The total phospholipids content was measured spectrophotometrically, using calibration curve, according to AOCS, 1989 .The soap content was determined volumetrically, according to AOCS, 1985 . The oilThe free fatty acid content was measured according to ISO 660, 2020 .The total carotenoids content  was determined by a spectrophotometric method  measurinThe oil color was determined measuring pure oil sample transparency (% T) at 455 nm using a UV/VIS spectrophotometer T80+ , as described Dimić and Turkulov, 2000 [3/H2O2 was provided as a blank sample to assess the contamination. The samples were digested according to the manufacturer’s recommendation (Milestone Ethos Microwave Digestion System method). After cooling at room temperature, the digests were diluted into a 25 mL plastic flask and transferred to vessel for further analysis. Quantification of Fe and Cu was conducted using acidified aqueous metal standards  by an external calibration procedure.The determination of heavy elements was performed by inductively coupled plasma mass spectrometry (ICP-MS). Approximately 0.5 g of the oil sample was measured and transferred into the Teflon vessels for microwave digestion. Thereafter, 8 mL of concentrated nitric acid ) and 1 mL of hydrogen peroxide ) were added. Microwave digestion system  was used for the digestion of the oil samples. A mixture of HNOVarious classical machine learning models are widely utilized in modeling across different scientific fields. These models include artificial neural network (ANN), random forest regression (RFR), support vector machine (SVM), extreme learning machine (ELM), K-nearest neighbors (KNN) and decision tree (DT). SVM, a discriminant technique based on statistical learning theory, is recognized for its exceptional generalization ability. By striking a balance between model complexity and training error, the optimal network is achieved . ELM conState-of-the-art machine learning techniques offer a diverse range of options for sequence data, such as ensemble learning models, such as XGBoost , LightGBIn this paper, artificial neural network model ,52, as aThe ANN modeling technique was chosen for prediction purposes due to its proven efficiency in approximating nonlinear functions ,54.The ANN model building structure was based on the multi-layer perceptron model (MLP) scheme, comprised of three layers .W1, B1, W2 and B2, with Y as the output variables matrix, f1 and f2 as activation functions in the hidden and output layers, and with X as the matrix of input variables [The MLP-formed ANN model could be presented using matrix notation, with weight and bias coefficients associated to the hidden and output layer written in matrices ariables :(1)Y=f1. Throughout the learning procedure, ANN inputs were supplied with a training set of parameters in order to establish the optimal number of neurons in the hidden layer to estimate the weights and bias coefficients and non-linear activation functions for every neuron in the ANN model.During the iterative process of calculating weights and biases coefficients and testing different activation functions for the hidden and output layers, the Broyden−Fletcher−Goldfarb−Shanno (BFGS) algorithm was employed. Various activation functions were explored, including hyperbolic tangent, logistic sigmoidal, exponential and identity functions. The identity function directly passes the activation level from the input as the output of the neurons. Logistic sigmoid uses the S-shaped logistic sigmoid function, producing output values in the range of 0 to +1. The hyperbolic tangent function (tanh) is another symmetric S-shaped (sigmoid) function, with output values ranging from −1 to +1. It often outperforms the logistic sigmoid function due to its symmetry. The exponential function utilizes the negative exponential activation function . A sequeYoon’s interpretation method was used to determine the relative influence of input variables on the removal efficiency of chemical parameters , oil transparency (at wavelength of 455 nm) and Fe and Cu content after filtration process . This mep < 0.05. Statistical processing of the obtained results and ANN modeling was performed using Statistica version 13.5.0.17 .The results were revealed by mean value ± standard deviation (n = 3). Data were analyzed by one-way analysis of variance (ANOVA) with a Tukey HSD test. It was used to determine significant differences at the significance level The investigation results of the content of waxes, moisture, phospholipids, soap, free fatty acids, iron, copper, total carotenoids content, as well as oil transparency before and after filtration are shown in 2 value during the training cycle was achieved when using nine hidden neurons for constructing the ANN model  technique was employed. The structure and outcomes of the ANN model depend on the initial assumptions of matrix parameters, which are crucial for building and fitting the ANN to experimental data. Moreover, the behavior of the ANN model can be influenced by the number of neurons in the hidden layer. To address this concern, each network topology was iterated 100,000 times to minimize random correlations caused by initial assumptions and random weight initialization. Through this approach, the highest rNN model a.The model underwent training for 100 epochs and 2 values were equal to 1.000, during the training cycle r2 for output variables, hidden and output layer activation functions were logistic sigmoid , oil transparency (at wavelength of 455 nm) and Fe and Cu content after filtration process , compare sigmoid .The developed ANN model consisted of 117 weights−bias coefficients due showing the high nonlinearity of the system ,60.2 values, while the sum of squares (SOS) values acquired using the ANN model were of the same order of magnitude as experimental errors for outputs mentioned in the literature [For the ANN model, the model-calculated values were not too close to the experimental values in most cases, in terms of rterature ,56.−1 waxes in refined oil [−1, while the lowest wax content was sample F_16 (281 ± 8 mg kg−1). According to the results shown in −1, respectively), while the lowest wax content values were 2.89 ± 0.04 and 2.89 ± 0.05 mg kg−1, found in F_09 and F_16 samples. Similar results were reported by Mitrović et al., 2009 [−1. The wax content in the winterized oil obtained by the turbidimetric method ranged from 5.91 mg kg−1 to 16.07 mg kg−1 [p = 0.00). The same conclusion applied to global sensitivity analysis. Namely, wax content in the initial samples was the most important parameter for the prediction of W_out, with relative importance of +33.40%  using a sodium hydroxide (NaOH) were removed in the form of soap. Obtained soaps are water soluble, so oil washing reduces soap content easily to minimum values, below 50 mg/kg , even beThe moisture content is an important quality indicator of crude and refined oils. The presence of moisture in the oil is undesirable for several reasons, primarily economic interest and quality issues . The moiFree fatty acids are formed by hydrolytic cleavage of triacylglycerol during inadequate storage and preparation of seeds for pressing and during pressing . The resCarotenoids are a large group of polyunsaturated hydrocarbons, composed of isoprene residues  and domiThe oil transparency values are a consequence of different pigment content, primarily total carotenoids. The total pigments content in the oil is inversely proportional to the oil transparency value . In the Traces of metal ions increase the oxidation degree of edible oils and fats, and thus affect the sensory characteristics of oils ,74. The Winterization process and oil filtration assisted by cellulose-based filtration aids were proven to be very effective in wax removal; namely, over 99% waxes were removed. According to the obtained results, filtration with filtration aids based on cellulose had multiple beneficial effects during filtration of sunflower oil; in addition to the wax content, the total phospholipid content (50–100%) and the soap content (100%) were also reduced.2 values during training cycle for these variables were 1.000). Results of the global sensitivity analysis showed that the quantity of tested cellulose filtration aids and the filtration time accelerated the removal of only some of the tested parameters, which opens the possibility of further testing of the combination of these aids. The experiments had been planned in a way that the waxes should have been removed from the oil almost completely, and that goal was achieved. Based on the obtained results, it was concluded that there is a possibility to change the quantity of added filtration aid and to achieve the desired result. Therefore, future plans are to carry out research on the optimization of the filtration aid quantity and filtration time, aiming to reduce the operational costs.The results of this study disclose the removal efficiency of chemical parameters , oil transparency and Fe and Cu content after filtration process, based on data of initial values of chemical parameters, oil transparency, Fe and Cu content, and also the data regarding filtration time and the quantity of filtration agent. The artificial neural network model was adequate for the prediction of output variables (the r


Oncorhynchus mykiss. The supplement has been shown to maintain the viability of fish affected by a natural bacterial infection as well as alter the fatty acid composition and decrease oxidative damage to the liver. Thus, plant-origin substances could be readily available and safe alternatives to pharmacotherapy and used as a way to improve the health of fish and promote their ability to tolerate stressors under intensive production.Cultivated fish face multiple stressors that impact their viability, growth, and health; additionally, environmental stressors provoke the transmission of bacterial diseases in fish populations. It is widely recognized that feed supplements of plant origin can alleviate stress in fish via the stimulation of non-specific defense responses. We tested the effects of a dietary mix consisting of an antioxidant, dihydroquercetin, and a prebiotic, arabinogalactan, on growth and biochemical indices in farmed rainbow trout, Oncorhynchus mykiss, received either a standard diet or a diet supplemented with 25 mg/kg of dihydroquercetin and 50 mg/kg of arabinogalactan during a feeding season, from June to November. The fish in the control and experimental groups were sampled twice a month  for growth variable estimations and tissue sampling. The hepatic antioxidant status was assessed via the quantification of molecular antioxidants, such as reduced glutathione and alpha-tocopherol rates, as well as the enzyme activity rates of peroxidase, catalase, and glutathione-S-transferase. The lipid and fatty acid compositions of the feed and fish liver were analyzed using thin-layer and high-performance liquid chromatography. The viability, size, and biochemical indices of the fish responded to the growth physiology, environmental variables such as the dissolved oxygen content and water temperature, and sporadic factors. Due to an outbreak of a natural bacterial infection in the fish stock followed by antibiotic treatment, a higher mortality rate was observed in the fish that received a standard diet compared to those fed supplemented feed. In the postinfection period, reduced dietary 18:2n-6 and 18:3n-3 fatty acid assimilation contents were detected in the fish that received the standard diet in contrast to the supplemented diet. By the end of the feeding season, an impaired antioxidant response, including reduced glutathione S-transferase activity and glutathione content, and a shift in the composition of membrane lipids, such as sterols, 18:1n-7 fatty acid, and phospholipids, were also revealed in fish fed the standard diet. Dietary supplementation with plant-origin substances, such as dihydroquercetin and arabinogalactan, decreases lethality in fish stocks, presumably though the stimulation of natural resistance in farmed fish, thereby increasing the economic efficacy during fish production. From the sustainable aquaculture perspective, natural additives also diminish the anthropogenic transformation of aquaculture-bearing water bodies and their ecosystems.Natural feed supplements have been shown to improve fish viability, health, and growth, and the ability to withstand multiple stressors related to intensive cultivation. We assumed that a dietary mix of plant-origin substances, such as dihydroquercetin, a flavonoid with antioxidative, anti-inflammatory, and antimicrobial properties, and arabinogalactan, a polysaccharide with immunomodulating activity, would promote fish stress resistance and expected it to have a protective effect against infectious diseases. Farmed rainbow trout fish,  Oncorhynchus mykiss, is one of the most cage-cultured fish species in high-latitude countries due to its fast growth in low-temperature environments [Larix gmelinii and L. sibirica) and is structurally similar to other bioactive flavonoids such as quercetin, hesperidin, or rutin. It is widely used in the medical and food industries due to its antioxidative, anti-inflammatory, antimicrobial, and antitumor activities [Rainbow trout, ronments ,2. Unlikronments . In orderonments ,5,6. Somtivities ,8,9,10. tivities  and supptivities  or no imtivities . Arabinotivities ,15,16,17tivities ,19,20 antivities ,22,23,24tivities ,26,27,28Oncorhynchus mykiss) that received either a standard diet or a diet supplemented with dihydroquercetin and arabinogalactan throughout a growing season for five months. Due to an episode of a natural bacterial infection that occurred during the observation period, the antioxidant and immunomodulating properties of a natural additive were able to be verified.The aim of the study was to estimate some physiological and biochemical indices in farmed rainbow trout (Oncorhynchus mykiss) yearlings  were placed in four cages with an initial stocking density of 2.1 kg/m3; the total biomass of the fish in each cage was around 886 kg. The fish were fed one of two diets (in duplicate), either the commercial diet BioMar  throughout the study period were between 6 and 18 °C and 5 and 11 mg/L, respectively . The watPseudomonas putida and Cytophaga psychrophile. Two weeks after the disease manifestations, the fish in all cages were treated with an antibiotic, enrofloxacin (25 mg/kg of fish weight), using a six-day regimen.During the observation period, a sporadic bacterial disease occurred in the fish stock, with the initial manifestation on 12 July in rainbow trout yearlings in both groups. The infected fish swam on their sides, refused food, and had hepatic abnormalities and hemorrhages in the liver. The infection was identified as a bacterial hemorrhagic septicemia-type disease caused by an association of 1). The liver samples were dissected with scissors, then frozen in liquid nitrogen and stored at −80 °C until the enzymatic assay and evaluation of reduced glutathione and vitamin contents. For the lipid analysis, hepatic samples were fixed in 97% ethanol (with the addition of 0.001% butylated oxytoluene as an antioxidant) and stored at +4 °C.At the indicated sampling dates, eight fish from each cage were euthanized with an overdose of clove oil , the cages were lifted from the depths to the count dead fish accumulated at the bottom of the cages. Thus, the November mortality values included the number of unnoticed dead fish accumulated at the bottom of the cages since August . The totO. mykiss were conducted in the Equipment Sharing Center of the Karelian Research Center of the Russian Academy of Sciences .Biochemical analyses of hepatic antioxidants and lipid profile in the rainbow trout g for 1 h at 4 °C in an Allegra 64R centrifuge . The resultant supernatant was used for a biochemical assay of the enzyme activities and GSH level.The frozen 0.1–0.4 g liver samples were homogenized with a Digital Disruptor Genie unit  in 5.0 mmol Tris-HCl buffer, pH 7.5. The homogenate was centrifuged at 60,000× The glutathione S-transferase (GST) activity was determined from the rate of reduced glutathione (GSH) binding with the 1-chloro-2,4-dinitrobenzene (CDNB) substrate . A microThe catalase (CAT) activity was determined using a technique by Beers and Sizer . The actThe peroxidase (Px) activity was determined using a technique used by Maehly and Chance  in a reag for 15 min. The supernatant was adjusted to pH 8.5 with 5 mol of NaOH and then mixed with 0.4 mol of Tris-HCl at pH 8.5, containing 5 mmol of ethylene diamine tetraacetic acid (EDTA). Then, 0.01% ortho-phthalaldehyde in methanol, prepared immediately before use, was added to the reaction mixture. The mixture was stirred and incubated at room temperature for 15 min, after which its fluorescence was measured . The reduced glutathione concentration was calculated according to the calibration curve plotted using series of solutions with different GSH concentrations in 0.4 mol of Tris-HCl at pH 8.5, containing 5 of mmol EDTA. The relative glutathione concentration was expressed in GSH µg per mg protein.The concentration of reduced glutathione (GSH) was determined based on the Cohn and Lyle method  modifiedThe soluble protein concentration in the supernatant was measured spectrophotometrically through peptide bond absorbance at 220 nm at 26 °C .g for 10 min. The hexane layer was injected onto the HPLC system [v/v) was applied as the mobile phase. The eluate was monitored at 292 nm, and the vitamin was identified by the retention time compared with the pure standard .The concentration of alpha-tocopherol (vitamin E) in the fish liver was determined using high-performance liquid chromatography (HPLC). The samples of tissues were homogenized in 0.25 mol of sucrose solution at pH 7.4 and mixed with an equal volume of 0.125 mg/mL of butylated oxytoluene in ethanol to precipitate the proteins. Next, each sample was diluted twice with n-hexane. The mixture was vortexed for 5 min for the extraction of vitamins and centrifuged at 3000× C system . Chromatv/v) according to Folch et al. [v/v) as the mobile phase. The identification of the fractions was performed using standards such as the phospholipid mixture , cholesterol , glyceryl trioleate , and cholesteryl palmitate . The quantitative composition of the fractions was measured at 540 nm for phospholipids, triacylglycerols, and cholesterol esters and at 550 nm for the sterol fraction using an SF-2000 UV/Vis spectrophotometer  [Lipids were extracted with chloroform/methanol  ,38.v/v) and a UV-spectrophotometer at 206 nm using a Stayer liquid chromatography system . Peaks were identified by referencing the retention times of the authentic standard phospholipid mixture  and phosphatidylserine .The composition of the phospholipid fractions, including phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), was determined by high-performance liquid chromatography using the method of Arduini et al.  on a NucFatty acid methyl esters (FAME) from the total lipids were prepared using methanol and acetyl chloride. The FAME were separated on an Agilent 7890A gas–liquid chromatograph  with a flame ionization detector using DB-23 columns (60 m–0.25 mm)  and nitrogen as the mobile phase. The FAME from fish total lipids were identified by comparison with standard mixes . Minor fatty acids constituting less than 1% of the total fatty acids were not identified.p-values adjusted using the Holm method. The weight gain rates in the two groups were compared using an ANCOVA. The differences were considered significant at p ≤ 0.05. The values of the studied biochemical parameters are presented as the median values ± half of the interquartile range.The statistical analyses were performed using R Statistical Software . DiffereDuring the observation period, a significant difference in fish mortality was observed between the studied fish groups . The totThe weight gain in the farmed trout of either group was linear , as well as with sterols and vaccenic 18:1n-7 and palmitoleic 16:1n-7 acids  A, indicaIn both studied fish groups, the TAGs were intensely accumulated in summer in accordance with the total fat deposition   and posiThe dietary supplementation with arabinogalactan and dihydroquercetin for just a week resulted in an increase in the 15:0 fatty acid and a decrease in the 18:0 fatty acid contents in fish livers A,B. TherClarias gariepinus [Oreochromis mossambicus [Dihydroquercetin- and arabinogalactan-containing feed had a beneficial effect on fish viability, significantly reducing the cumulative mortality rate within the growing season. The impaired welfare observed in the supplement-free fish group was likely a result of a more severe infection, lower susceptibility to the antibiotic treatment, re-infestation, or other causes. The similar growth rates mean that regardless of the diet, the fish consumed food and effectively converted the obtained energy into body mass. The unperturbed growth but lower viability in fish that were fed a standard diet possibly indicate the sudden death of individuals due to sporadic acute infection rather than the chronic effect of any undetermined hazardous agents on caged fish. We assume that the tested supplement mixture may have a beneficial long-lasting effect on fish viability, probably via increasing the resistance to both infections and re-infections. Our data on the influence of the studied plant-origin substances on the survival and welfare of rainbow trout correspond with data in the literature describing the effects of other related compounds on the welfare and mortality of fish. For example, medicinal plant feed additives enhanced the growth and survival of cultured riepinus . Dietarysambicus .A number of stress factors  initiating reactive oxygen species (ROS) generation and lipid peroxidation (LPO), and consequently providing oxidative stress, affect the survival and growth of trout ,44,45,46O. mykiss liver are membrane phospholipids. Together with sterols (mainly cholesterol), they comprise cell membranes and affect membrane fluidity and the activity of membrane-bound enzymes, ion channels, and receptors [Being a key component of various metabolic pathways, lipids play a crucial role in the survival and tolerance of an organism to environmental stressors. Cell lipids contain polyunsaturated fatty acids enriched by double bonds within their hydrocarbon chains, which are primary targeted by pro-oxidants and can indicate LPO progression. The main lipid components of the eceptors ,53,54. Meceptors ,56. PhosThe water temperature determines the dissolved oxygen content and feed consumption rate of the fish and eventually their metabolic rate , and dirTriacylglycerols (TAGs) are high-energy lipid molecules stored during intense feeding, readily covering the energy costs needed to maintain homeostasis at different life stages or in different environments ,56. In bAccording to previous observations, the fatty acid composition of the fish liver is very similar to that of commercial feed, with an equal ratio of oleic 18:1n-9, linoleic 18:2n-6, alpha-linolenic 18:3n-3, gamma-linolenic 18:3n-6, eicosapentaenoic 20:5n-3, and palmitoleic 16:1n-7 acids, suggesting their common exogenous origin ,58,59,60The accumulation of 15:0 fatty acid  and a decrease in 18:0 coincided with the initial stage of infection, and this response may arise from exposure to stress or pathogens. Although 15:0 acid is known to be of bacterial origin, it still unclear whether it was sourced from pathogenic or symbiotic microbiota or if these components were unequally assimilated from food by the fish in both studied groups .Since linoleic and alpha-linolenic acids are both essential and food-derived, these results can be interpreted as an impaired digestion sign in supplement-free fish. The beneficial effect of dietary arabinogalactan on symbiotic intestinal microflora (bifidobacteria and lactobacilli) as well as the boosting of immunological characteristics were reported previously in mammals . The acc® (including dihydroquercetin), a licensed drug against liver cirrhosis [Sterols (mainly consisting of cholesterol) and phosphatidylethanolamine both comprise important indexes of membrane fluidity, such as the ratios of phospholipids to cholesterol and phosphatidylcholine to phosphatidylethanolamine . The regirrhosis . It shouirrhosis ,66. HoweDue to the poor response of the antioxidant system and moderate loss of lipid substrates for oxidation during the bacterial infection, the protective effect of dihydroquercetin and arabinogalactan against severe oxidative stress ,44,45 inO. mykiss, were revealed. The welfare of the fish in both groups was impacted by a sporadic bacterial infection, including an increased mortality rate and impaired assimilation of nutrients, particularly the malabsorption of essential linoleic 18:2n-6 and alpha-linolenic 18:3n-3 fatty acids. The feed supplementation partially reduced both the lethality of the fish stock and the impairment of their digestion during the infection and at the postinfection period. Therefore, the enhanced resistance to the infection and an accelerated postinfection recovery rate observed in fish fed a supplemented diet could be the results of the biological activity of plant substances. Presumably, plant-origin substances stimulate vitality and natural resistance in farmed fish though increasing the efficacy of fish production. Although the benefits of these feed supplements in fish production are indisputable, further experiments under controlled conditions are required to study the mechanism of the immunomodulating activity of the dietary mix in detail.Some beneficial effects of the dietary mix of dihydroquercetin and arabinogalactan on physiology responses of farmed rainbow trout,


Increasing women’s health literacy is the key to preventing cervical cancer, and various tools have been developed to assess women’s cancer health literacy. However, many of these tools come from other countries and have not been adapted to Chinese requirements. Furthermore, a system for evaluating cervical cancer health literacy among Chinese women has not been developed. Therefore, we sought to establish an evaluation index system for cervical cancer health literacy among Chinese women and to provide an effective evaluation tool for tertiary prevention of cervical cancer in China.We invited 20 recognized experts to participate in two rounds of Delphi expert consultation, and the modified Delphi process with percentage weighting and multiplication was used. A literature review identified 67 potential indicators. Subsequent discussions within our research team led to the retention of 48 indicators following a rigorous screening process. On this basis, two rounds of Delphi expert consultation were conducted to rate and screen the indexes. Percentage weighting and multiplication were used to determine index weights.W coefficients ranged from 0.168 to 0.248. The weights of first-level indicators of basic knowledge and attitudes about cervical cancer, primary prevention of cervical cancer literacy, secondary prevention of cervical cancer literacy, and tertiary prevention of cervical cancer literacy were 0.257, 0.249, 0.251, and 0.243, respectively.Twenty experts participated in the first-round Delphi consultations (95.23% recovery rate). In the second-round Delphi consultations, 20 questionnaires were returned (100%), and the expert authority coefficient was 0.93 ± 0.02. After both rounds of Delphi consultation, 4 first-level indicators, 9 second-level indicators, and 32 third-level indicators were identified for cervical cancer literacy among Chinese women. On a five-point scale, importance ratings ranged from 3.76 to 4.95 points, with variation coefficients ranging from 0.06 to 0.25, while sensitivity ratings ranged from 3.71 to 4.83 points, with variation coefficients ranging from 0.08 to 0.24. Across both rounds, Kendall’s We have developed the first tertiary prevention-based, comprehensive evaluation index system for cervical cancer literacy among Chinese women, which will provide theoretical support for cervical cancer prevention and health education programs.The online version contains supplementary material available at 10.1186/s12885-023-11208-3. Cervical cancer is the fourth-most prevalent cancer among women worldwide. It is a significant public health issue, resulting in approximately 604,000 new cases and 342,000 deaths in 2020 .MoreoverHuman papillomavirus (HPV) infection causes 90% percent of cervical cancers. Thus, the adoption of primary and secondary prevention methods can effectively prevent cervical cancer , 6. The The concept of cervical cancer health literacy refers to individuals’ capacity to obtain, process, and comprehend basic health information and services that will enable them to make appropriate health decisions. Improving cervical cancer health literacy levels is one of the keys to preventing cervical cancer . SeveralTo identify potential cervical cancer health literacy indicators, a comprehensive literature search was conducted in various databases, including PubMed, Web of Science, China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database, and Wanfang Data. The search covered articles published from January 1, 2020, to December 31, 2021. The retrieval strategy employed the following:  *) *(HPV vaccine [Title])) *(HPV [Title])) *(human papillomavirus [Title])) *) and )) + ) *)) and (tertiary prevention [[Title/Abstract]). After assessing the abstracts and eliminating irrelevant articles, 270 references were considered for further analysis  information about the experts, such as their gender, age, and years of experience; (2) content of the Delphi expert consultation, where experts rated each indicator in terms of importance and sensitivity using a Likert 5-level scoring method with 1–5 points indicating answers ranging from “very unimportant/sensitive” to “very important/sensitive”; and (3) familiarity scale with cervical cancer health literacy on a scale of 1 (very unfamiliar) to 5 (very familiar) points. Additionally, experts were consulted about each indicator’s deletions and additions and their remarks were included in a comments column.To meet the minimum requirement of 15 experts as stipulated by the Delphi expert consultation method, we initially planned to select a panel of 15–20 experts based on specific circumstances. To ensure the expertise and credibility of the experts and to consider the regional disparities in cervical cancer prevention and control, we randomly invited experts from provincial-level maternal and child health hospitals, disease control centers, health bureaus, and health education centers across China. Ultimately, we selected a diverse group of twenty experts from various regions, encompassing specialties such as women’s health, cervical cancer diagnosis and treatment research, health education, and disease prevention. Experts who participated in the Delphi consultations were required to (1) have worked in their respective field for more than 10 years, (2) be familiar with the research topic, and (3) be able to provide comprehensive opinions and participate enthusiastically in both rounds of consultation.In June and September 2022, Delphi expert consultations were conducted via email. In the first round, experts ranked each potential indicator’s importance and sensitivity on a five-point Likert scale. To ensure that the experts understood each indicator, they were defined in detail. Experts could also make comments about each indicator or suggest adding or removing specific indicators. Using indicator screening criteria, we collected open opinions on the indicators in the first round, deleted and revised them, and sent them for the second round of expert consultations. Experts’ scores on the importance and sensitivity of each indicator were collected again during the second round of expert consultations, and the final indicator system was created. Similarly, the same 20 experts were invited to complete the questionnaires in the second round.Screening criteria included an average score of > 3.50 points for indicator importance and sensitivity and a coefficient of variation of < 0.25 as the quantitative standard for indicator retention. If the indicators did not meet the above criteria or if the experts recommended adding, modifying, or deleting them, the research team made a final decision after discussing and considering all suggestions .After the Delphi consultation, the percentage weighting method was used to calculate the weight of the primary indicators, and the percentage weight method combined with the production method was used to calculate the weights and combined weights of the secondary and tertiary indicators. First, the importance of each indicator was rated (1–5 points) by experts during the second round of consultations, and the average value was calculated. To determine the weight of the primary indicator, the average score of that indicator was divided by the sum of all the primary indicators’ average scores. A secondary index’s percentage weight was calculated by multiplying its percentage weight by its weight in the primary index it belonged to, using the same primary index as a whole. As a result, the combined weight of the secondary index was obtained. Finally, we calculated the weight and combined weight of the tertiary index .W coefficient of concordance of the experts’ evaluation of the importance and sensitivity of the indicators during both rounds of consultations.SPSS version 26.0  and Excel 2010  were used to calculate the mean, standard deviation, and coefficient of variation values of each index’s importance and sensitivity scores, as well as the expert’s positive coefficient, expert’s authority coefficient, and weights of indicators at all levels. SPSS version 26.0 was used to calculate the Kendall The first-round Delphi consultation involved 20 experts (the recovery rate was 95.23%). For the second round of Delphi consultation, revised questionnaires were sent to the same 20 experts, and a total of 20 questionnaires were returned (for a recovery rate of 100.00%). As shown in Table An additional 1 shows the concentration and variation of expert opinions. According to the first round, the mean importance scores for potential indicators ranged from 3.79 to 4.89 points, with variation coefficients ranging from 0.06 to 0.25, while the mean sensitivity scores ranged between 3.74 and 4.68 points, with variation coefficients ranging from 0.11 to 0.28. Following the second round, potential indicators scored between 3.76 and 4.95 points on the importance scale, with variation coefficients varying from 0.04 to 0.24, while the potential indicators’ sensitivity scores ranged between 3.71 and 4.83, with variation coefficients ranging from 0.08 to 0.24.W coefficients ranged between 0.168 and 0.248 in both rounds, and the importance and sensitivity scores in both rounds were all effective (p < 0.01), suggesting consistency among experts.The coordination of experts’ opinions is shown in Table In the first round of the study, three third-level indicators  were deleted, and two third-level indicators were revised . As a result, 4 first-level, 9 second-level, and 32 third-level indicators were included in the second round of consultation. Then, as part of the second round of revisions, a third-level indicator was revised . Ultimately, there were 4 first-level indicators, 9 second-level indicators, and 32 third-level indicators  received the highest score, which indicates that women need to be educated on cervical cancer screening, early detection, and the symptoms of cervical cancer. Meanwhile, cervical cancer tertiary prevention literacy received the lowest weighted indicator (0.243), which indicates that experts believe that knowledge regarding seeking timely medical care after a cervical cancer diagnosis is relatively less important for the average woman. This is in line with the needs of health education work and suggests that cervical cancer patients could be the focus of this part of the work.The assessment indicators prioritized tertiary prevention to reduce cervical cancer incidence and mortality in Chinese women. It identified knowledge gaps in cervical cancer prevention and provided targeted educational materials and interventions to increase awareness of cervical cancer and its risk factors. This evaluation indicator can give a more comprehensive assessment of cervical cancer health knowledge among Chinese women. It can help policymakers assess the effectiveness of prevention efforts, identify areas of low ability, and inform targeted policies and interventions. Collaboration between researchers, healthcare providers, and policymakers is crucial for effectively implementing the evaluation system in China’s cervical cancer prevention and health education programs. We suggested integrating the system with existing prevention efforts, using culturally appropriate indicators, and continuously evaluating program effectiveness. This study developed an indicator system suitable for Chinese women for cervical cancer health literacy by fully incorporating existing knowledge indicators for tertiary prevention of cervical cancer. We conducted an extensive literature review and expert interviews to ensure comprehensiveness and representativeness. The inclusion of experts in various fields of cervical cancer prevention assured the authority and validity of the Delphi survey data. However, as Chinese women lack knowledge about cervical cancer prevention, we did not interview them for cervical cancer health knowledge indicators. Instead, experts were asked to select indicators from the perspective of the average Chinese woman. Although the indicator system has not yet been implemented in a large sample of Chinese women, we plan to validate its reliability and validity in a follow-up study.In our study, following the Delphi method strictly, we reached a good consensus on 45 indicators and determined the weights of indicators at all levels using percentage weights and multiplication. This is also the first study to develop a tertiary prevention-based, comprehensive evaluation index system for cervical cancer literacy among Chinese women, which will provide theoretical support for cervical cancer prevention and health education programs.Below is the link to the electronic supplementary material.Additional file 1Additional file 2


Recent studies have unveiled an association between socioeconomic position (SEP) and intrahepatic lipid (IHL) content. The aim of this study was to examine to what extent traditional lifestyle factors mediate the relationship between SEP and IHL content, independent of aetiology, and non-alcoholic fatty liver disease (NAFLD).We used cross-sectional data derived from The Maastricht Study . Education, income, and occupation were used as indicators of SEP. Physical activity (accelerometer), intake of total energy, alcohol, saturated fat, protein, vitamin E, dietary fibre, and fructose from sugar-sweetened beverages (SSBs) and fruit juice (food frequency questionnaires) were potential mediators. IHL content was quantified by magnetic resonance imaging. Age, sex, and type 2 diabetes were covariates. Multiple parallel mediation analyses  were performed.Individuals with a low education level had a 1.056-fold higher IHL content (95% CI: 1.03–1.08) and a 44% greater NAFLD risk  compared with those with higher education levels. Approximately 8.9% of educational disparity in risk of IHL content was attributable to moderate-to-vigorous physical activity; 6.3% to fructose intake from SSBs; 5.5% to dietary fibre; and -23% to alcohol. Approximately 8.7% of educational disparity in risk of NAFLD was attributable to moderate-to-vigorous physical activity; and 7.7% to fructose intake from SSBs. However, the indirect effect of these mediators was small (0.998 for IHL content and 1.045 for NAFLD) in comparison to the total effect. Similar results were found when income and occupation were used as SEP indicators.Societal measures may alleviate the burden of NAFLD and further studies that identify mediators other than traditional lifestyle factors are warranted to define the relationship underlying SEP and IHL content.Individuals with a low or medium level of education, income, or occupational status had more fat accumulation in their livers than individuals with a higher education, income, or occupational status. This difference may be attributed to the influence of unhealthy lifestyle factors, such as reduced physical activity and a higher intake of sugar-sweetened beverages among individuals with lower socioeconomic position. Nevertheless, other yet unknown factors may also play a role.  •Low socioeconomic status was associated with a higher IHL content and a greater risk of NAFLD.•Physical activity, dietary fibre, and fructose intake from sugar-sweetened beverages were significant mediators of this effect.•Traditional lifestyle factors only partly mediated the relationship of socioeconomic position with IHL content and NAFLD. Approximately 32.4% of adults,,,Many studies have identified lifestyle factors in the pathogenesis and progression of NAFLD. These factors include excess caloric intake,Interventions focusing on modifying lifestyle factors to treat and prevent the accumulation of IHL and the onset of NAFLD requires a better understanding of the factors that characterise an unhealthy lifestyle and how they relate with IHL content and NAFLD. Although many studies have examined the relationship between a low socioeconomic position (SEP) and an unhealthy lifestyle,Therefore, in the present study, we examined the relationship between three SEP indicators  and IHL accumulation, independent of aetiology (as the primary outcome), and NAFLD , and assessed to what extent these relationships were influenced by traditional lifestyle factors.This study was conducted in accordance with the Guideline for Reporting Mediation Analyses (AGReMA) statement,We used data from the Maastricht Study, a population-based cohort study enriched with type 2 diabetes patients (T2D).i.e. the education dataset   measurements of the liver were performed from December 2013 onwards as part of the initial baseline workup and partly as a catch-up measurement . As many individuals had missing data on income  and occupational status , we constructed three separate datasets for each SEP indicator, = 4,001) , the inc= 4,001) , and the= 4,001) .The Maastricht study was approved by the institutional medical ethics committee (NL31329.068.10) and the Ministry of Health, Welfare and Sport of the Netherlands (permit 131088-105234-PG). All participants provided their written informed consent.In this study, self-reported information on educational, income, and occupational levels was used as SEP indicators. The educational level was reported through 9 ordinal categories: 1) no education, 2) uncompleted primary education, 3) primary education, 4) lower vocational education, 5) intermediate secondary education, 6) intermediate vocational education, 7) higher secondary education, 8) higher vocational education, and 9) advanced university education. For this study, three categories were created to define the educational level: low (1 to 4), medium (5 to 7), and high (8 and 9).Income was measured by self-reported net household income per month, and consisted of 19 categories, ranging from <€750 to >€5,000 per month. To compute equivalent income levels, household income was divided by the square root of household size. This implies that, for instance, a household of four persons has twice the needs of a single-person household.Participants were asked to describe their current or previous job. The job descriptions were then classified according to International Standard Classification of Occupations 2008 (ISCO-08), which is a hierarchical classification system based on education and skills required for a job.The inclusion of lifestyle factors as potential mediators of the relationship between SEP in IHL content was based on previously reported associations between individual lifestyle factors and IHL content.[9]Dietary intake was assessed using a tailor-made food frequency questionnaire (FFQ) developed using the Dutch national FFQ tool.Daily intake of total energy , alcohol (g/day), saturated fat (g/day), protein (g/day), vitamin E (mg/day), dietary fibre (g/day), fructose from fruit juice (g/day), and fructose from SSB (including sugar-containing fruit drinks and syrups) (g/day) were included as potential nutritional mediators.Physical activity was measured using a thigh-worn accelerometer  worn for eight consecutive days.IHL content was assessed by Dixon-MRI using a 3.0 T MRI system  with body matrix and supine radiofrequency coils.1H-MRS), the gold standard for non-invasive quantification of IHL, in 36 participants. After calibration, the intra-class correlation coefficient between Dixon-MRI and 1H-MRS was 0.989 (95% CI: 0.979–0.994).2/H2O ( × 100%).This method was validated and calibrated against proton magnetic resonance spectroscopy (2 (H2O + CH2), corresponds to 5.89% when IHL content is expressed as CH2/H2O, as was done in the present study.NAFLD is defined by the presence of hepatic steatosis with an IHL content ≥5.56%2). C-reactive protein, HbA1c, and lipid profiles were measured in venous blood samples.All participants completed questionnaires regarding age, sex, and history of cardiovascular disease (CVD). Medication use was assessed during medication interviews. Height, weight, waist circumference, and blood pressure were measured during a physical examination. BMI was calculated as kilogram per meter squared , which was defined according to the World Health Organisation 2006 criteria as normal glucose metabolism (NGM), impaired fasting glucose, and impaired glucose tolerance (combined as prediabetes status) and T2D.2 calculator v.2.2.3 for Windows.Insulin resistance was assessed by the homeostasis model assessment (HOMA-IR), which was calculated with the HOMAi.e. education level, income level and occupational level) (exposure) and all individual lifestyle factors (mediators) using linear regression. To fulfil the assumption of normality of linear regression, all lifestyle factors were transformed into square root values.Continuous data are presented as mean ± standard deviation (SD), or as median (IQR) in case of non-normal distribution of values. Categorical data are presented as number (%). All nutritional variables were adjusted for total energy intake by the residual method.10 transformed to fulfil the assumption of normality for linear regression. To obtain interpretable results we back-transformed the regression coefficients, which should be interpreted as the fold change (and not the additive change) in IHL content that is associated with one unit increase in certain lifestyle factor.Second, we conducted multivariable linear regression analysis to examine the association between lifestyle factors (mediators) and IHL content (outcomes). IHL content was log10 transformed, and the total effect, direct effect and indirect effect were then back-transformed. The high SEP indicator group was used as a reference  to quantify to what extent the association between the SEP indicator and IHL content is mediated by each lifestyle factor. For consistence of interpretation, IHL content was also logeference . All anaSeveral sensitivity analyses were performed. Mediation analyses were repeated after: 1) stratification by the MRI lag time (the time between basic measurements and the MRI of the liver); 2) excluding participants with T2D; 3) replacing ordinal SEP indicators by continuous SEP indicators; 4) replacement of MVPA by total physical activity; and 5) replacement of IHL content by NAFLD . Alcohol intake was excluded as a mediator in this analysis; 6) replacement of continuous mediators by binary mediators. The cut-off values for MVPA and nutritional mediators were derived from The Health Council of The Netherlandsp value of <0.05 was considered statistically significant in all analyses, except for interaction tests where a less stringent significance threshold of p <0.10 was applied.Statistical analyses were performed using R statistical software v4.0.1 with the bruceR packageBaseline characteristics of the overall population in the education dataset  were stratified according to IHL tertiles in The mean age of the study population was 60 ± 9 years, 49% were female, 20% were diagnosed with T2D, 21% were diagnosed with NAFLD, and the median IHL content was 3.2% (IQR: 2.0%–6.1%). The proportion of participants with excessive alcohol intake was 11% of women and 16% of men. Compared with participants in the lowest IHL tertile, those in the highest were older and more often male, had a lower educational level and less MVPA. Intake of total energy, alcohol, saturated fat, and fructose from SSB were higher, while intake of dietary fibre were lower in the highest IHL tertile when compared with individuals in the lowest IHL tertile.Participants in the highest IHL tertile were metabolically more unhealthy than participants in the lowest IHL tertile, as suggested by the lower HDL cholesterol, and higher BMI, serum triglycerides, HbA1c, HOMA-IR, c-creative protein, systolic and diastolic blood pressure, and prevalence of prediabetes, T2D and NAFLD. Moreover, the prevalence of CVD and the use of medication (including lipid-modifying and antihypertensive medication) were higher in the highest IHL tertile.Age, sex, and T2D adjusted analyses revealed that both individuals with low and medium education levels were characterised by a 5.6%- and 2.6%-fold higher IHL content, independent of the underlying aetiology, compared with individuals with higher education levels . These associations were stronger in women than in men  and IHL content, with a proportion-mediated effect of 8.9%, 5.5%, and 6.3%, respectively (vs. higher education)  . Of noteucation) .Fig. 1Mei.e. the time between the basic measurements and the MRI), the suppressive effect of alcohol was less pronounced in women  showed that individuals with low and medium education were characterised by a 44.3% and 44.8 % higher risk of NAFLD than individuals with a higher education . Mediation analyses demonstrated similar trends, although dietary fibre intake was no longer statistically significant . ReplaceBaseline characteristics of the participants in the income dataset  are shown in The associations between income level with lifestyle factors and lifestyle factors with IHL content are presented in vs. high income) and IHL content, with a proportion-mediated effect of 37.9% and 29.8%, respectively  showed that individuals with low and medium income were characterised by a 52.8% and 44.2% higher risk of NAFLD compared with individuals with higher income . The mediators were essentially similar . Replacep for interaction: 0.002 and 0.201, respectively; Baseline characteristics of the participants in the occupation dataset  are shown in The associations of occupational level with lifestyle factors and lifestyle factors with IHL content are presented in vs. high occupational level) and IHL content, with a proportion-mediated effect of -25.1%  showed that individuals with low and medium occupational levels were characterised by a 47.5% and 36.0% higher risk of NAFLD compared with individuals with a higher occupational level . Mediation analyses showed similar results . Replacevs. high education) and IHL content, whereas alcohol intake was a statistically significant suppressor.In this study, we found that individuals with low and medium SEP had a higher IHL content, independent of aetiology, and had a greater risk of NAFLD compared with individuals with high SEP. These associations were stronger in women than in men. MVPA, and dietary intake of fibre and fructose from SSB were significant mediators of the association between low education  and IHL content, which may explain the non-significant relationship between income and IHL content. We subsequently narrowed our analysis by focusing on NAFLD as the secondary outcome, which is solely attributable to metabolic risk abnormalities.To date, only one study has addressed the relationships between SEP, dietary intake, physical activity and NAFLD at the population level. That study reported that high education, but not household income was associated with a reduced risk of NAFLD. This association was partially mediated by high quality diet and increased physical activity.,,The interpretation of the current findings is complex, as educational level does not reflect health literacy alone, as income level is not the only indicator of financial resources. For instance, studies have shown that both adults and children correctly rate SSBs as the most unhealthy beverages.e.g. subsidised physical activity and levies on SSBs, which have been shown to be effectivee.g. enhancement of neighbourhood walkabilityThe implications of this study are severalfold. First, it illustrates the complex pathogenesis of NAFLD. The global prevalence of NAFLD has risen to epidemic proportions, i.e. the relationship between SEP and IHL not accounted for by the mediators under investigation, was even greater than the total effect. This may be attributed to the fact that the net mediating effect of traditional lifestyle factors (= indirect effect) was negative due to the substantial suppressive effects of alcohol. This implicates the existence of other, yet unmeasured mediators. Future studies are warranted to unveil the role of other putative mediators, including exposure to endocrine disruptors  and air pollution, which both have been associated with both low SEP and NAFLD,Second, and equally important, our study convincingly showed that a substantial degree of the relationship between SEP and IHL content was not explained by the traditional lifestyle factors. We found that the direct effectThe study has several strengths and limitations. First, we used data from a large population-based cohort that was extensively phenotyped using state-of-the-art methods. This allowed for an accurate estimation of IHL content. Second, by using both IHL content, a continuous trait, and NAFLD, a binary trait, in our primary and secondary analyses, respectively, we show that our results are relevant and robust. Third, as mentioned before, instead of performing simple causal mediation analyses, we applied multiple parallel mediation analysis, which can evaluate a host of indirect effects by multiple mediators, revealing the complex mechanisms between SEP, lifestyle factors, and IHL content by decomposing the total effects. Indirect effects of different mediators reflect the importance of different pathways and, therefore, provide useful information for tailored policy making. Nonetheless, our study also has specific limitations. First, SEP and dietary intake were based on self-reported information and, hence, can be prone to bias. Moreover, although the FFQ has been validated against 24-h dietary recalls for intakes of mono- and disaccharides, fruit and drinks, it has not been validated for SSB.In conclusion, this population-based study demonstrates that MVPA, alcohol consumption, dietary fibre, and fructose intake from SSB are mediators/suppressors of the association between low SEP and IHL accumulation, independent of aetiology. These findings suggest that societal measures to alleviate the global burden of NAFLD are warranted. Furthermore, our observations also suggest that lifestyle improvement in patients with low SEP will not normalise their risk, since a substantial proportion of SEP factors, IHL content, and NAFLD incidence could not be associated with traditional lifestyle mediators. Further studies are needed to identify other mediators that may explain the relationship between SEP and IHL content.10.13039/501100004725Ministry of Economic Affairs (grant 31O.041), 10.13039/501100009709Stichting De Weijerhorst , the Pearl String Initiative Diabetes , School for Cardiovascular Diseases , 10.13039/501100011095Care and Public Health Research Institute , School for Nutrition and Translational Research in Metabolism , Stichting Annadal , 10.13039/100016244Health Foundation Limburg , and by unrestricted grants from JanssenCilag B.V. , Novo Nordisk Farma B.V. , Sanofi-Aventis Netherlands B.V. , and Medtronic .ZR was supported by Chinese Scholarship Council. The Maastricht Study was supported by the European Regional Development Fund via OP-Zuid, the Province of Limburg, the Dutch Concept and design of the study: ZR, MCGJB, HB, and CDAS. Generation, collection, assembly, analysis and/or interpretation of data and drafting the manuscript: ZR and MCGJB. All authors contributed to the intellectual content of the manuscript and review of the manuscript and approved the final version for submission. MCGJB is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Data are available from The Maastricht Study for any researcher who meets the criteria for access to confidential data; the corresponding author may be contacted to request data.All authors declare no conflicts of interest related to this manuscript.Please refer to the accompanying ICMJE disclosure forms for further details.


Background and Objectives: A growing number of epidemiological studies have suggested that diabetes mellitus may increase cancer risk and is implicated in numerous other metabolic and inflammatory disorders. The increase in proinflammatory cytokines plays a major role in insulin resistance and leads to hypoalbuminemia and micro- and macrovascular diabetes complications, including kidney disease and anemia. This study aimed to investigate the utility of carcinoembryonic antigen (CEA), C-reactive protein (CRP), serum albumin level, hemoglobin, and lactate dehydrogenase (LDH) as biomarkers for cancer risk, and the biological implications of diabetes on the evolution and prognosis of oncological patients. Material and Methods: We conducted a retrospective, longitudinal, observational study on a total group of 434 patients, of which 217 were diagnosed with a form of cancer and type two diabetes as a comorbidity, and the other 217 were a control group without diabetes. These patients were admitted to the oncology clinic. In subgroups, the same number of patients was considered, depending on the location of the oncological pathology. Anemia, hypoalbuminemia, elevated lactate dehydrogenase, glycated hemoglobin, and C-reactive protein levels are more pronounced in subjects with type two diabetes and cancer. Conclusions: The presence of diabetes negatively affects the clinical and biological prognosis of cancer patients.  A growing number of epidemiological studies have found that diabetes mellitus increases the risk of developing many types of cancer . The assOne study of 47 patients with diabetes and colorectal cancer showed an average HbA1c value of 6.0%, while 45% of them had an HbA1c of at least 7% [Long-term type two diabetes mellitus is associated with about 1.5 times increased risk of pancreatic cancer . A causaDiabetes was associated with a significantly more pronounced risk of lung cancer compared with patients without diabetes when the analysis was restricted to studies that accounted for smoking status. In contrast, this association disappeared when the analysis was restricted to studies that did not consider smoking status . SubjectChronic hyperlactatemia maintained by increased lactate formation from adipocytes in obese individuals has been found to precede the onset of diabetes  and mighLactic dehydrogenase prognostic value in patients with lung cancer was investigated by some researchers, but the findings were inconclusive. According to some studies, patients with lung cancer have worse prognoses when their LDH levels are higher. Some researchers discovered that this correlation was insignificant . Higher Anemia is the most common hematologic change in patients with malignancies . It may Several studies have suggested the importance of inflammatory biomarkers in the onset of diabetes and the monitoring of antidiabetic treatment, without necessarily being related to a form of cancer. Thus, elevated C-reactive protein levels have been associated with an increased risk of the development of diabetes , and LDHThe objectives of the study were to assess the utility of CEA, CRP, serum albumin level, hemoglobin, and LDH as biomarkers of cancer risk and the biological implications of diabetes on the evolution and prognosis of oncological patients.We performed a retrospective, longitudinal, observational study over a period of 2 years between 2016 and 2017, on two equal groups of patients, with 217 already having type two diabetes and being recently diagnosed with breast, lung, colorectal, or pancreatic cancer (the most frequent types of tumors associated with type two diabetes), and the other 217 without diabetes. All of them were hospitalized in the Oncology Medical Clinic of the Clinical County Emergency Hospital Craiova, Romania.Criteria for inclusion: patients with a maximum performance status of two, hospitalized in the Oncology Medical Clinic of Emergency Clinical County Hospital Craiova (Romania) at the initiation of oncologic treatment, with the diagnosis of lung, breast, colon, or pancreatic cancer, who signed an informed consent. These patients were divided into two subgroups: subgroup 1 with type two diabetes, and subgroup 2 without diabetes.Criteria for exclusion: both patients with other types of tumors, and patients with other types of inflammatory, acute, or chronic conditions, were excluded; also, patients for whom insufficient data were recorded or who did not sign an informed consent.The study group was represented by 217 patients, already with type two diabetes, who were hospitalized in the Oncology Medical Clinic of the Emergency Clinical County Hospital Craiova (ECCHC), in the period of 2016–2017, with a performance status of a maximum of two, to begin oncologic specific treatment: 81 patients with lung cancer, 45 patients with colon cancer, 38 patients with pancreatic cancer, and 53 patients with breast cancer. After that, another equal group was formed, composed of 217 patients without type two diabetes. All data, for all 434 patients, were collected from the medical records of patients hospitalized in the Oncology Medical Clinic of ECCHC.In this study, at the first admission, we evaluated the values of hemoglobin, CRP, HbA1c, LDH, alkaline phosphatase (FAL), serum albumin, and CEA in subgroup 1, with diabetes, and we compared them with the values found in subgroup 2, without diabetes. The HbA1c values in subgroup 2 were less than 5.7%, so type two diabetes and prediabetes were excluded [The following Emergency Clinical County Hospital of Craiova analyzers were used in the study: COULTER DXH for hemoglobin, MINDRAY BS 800 for CRP and LDH levels, ARCHITECT C8000 for serum albumin, and COBAS E411-2 for CEA and HbA1c.p-value < 0.05 being considered statistically significant. A confidence coefficient was also used, with an ‘r’ value close to 1 being considered a positive correlation.Statistical analysis was performed using Microsoft Office Pack - Microsoft Excel 2000 to collect data and obtain graphics and average values, and the MATLAB v.9.0 (2016) Student T-Test program to test the normality of data and expose the statistically significant differences between the two analyzed groups. The average values in the study group are compared with those of patients in the group without diabetes, a Ethical concerns: the protocol of the study was approved by the Ethics Committee of the University of Medicine and Pharmacy of Craiova, approval number 39/20.01.2023.p-value result was 0.002.The average values in the study group, compared with those of patients in the group without diabetes, were strongly correlated, and the p = 0.0001)  . The norp = 0.0033) (Lactic dehydrogenase studied in the entire group was more abundant in the diabetes subgroup at cancer diagnosis (average 337 U/L) versus the group without diabetes (292 U/L) ( 0.0033) .Glycated hemoglobin (HbA1c) values in subgroup 1 had rates between 5.7% and 13.1%, with an average value of 8.57%, which suggests poor diabetes control in relation to neoplasia. Non-diabetic patients were considered those with HbA1c values lower than 5.7%.p < 0.001)  .A positive correlation in the entire analyzed group was identified between LDH values and serum albumin values (r = 0.76), but also between LDH and hemoglobin values (r = 0.82), especially in those patients diagnosed in stage IV . Also, ap < 0.001)  .Also, a positive correlation between hemoglobin and C-reactive protein was found .Also, correlations between LDH and CRP levels (r = 0.83), and LDH and FAL (r = 0.72), were found. All these correlations suggest a sustained inflammatory response of the body in the presence of type two diabetes, more pronounced as the body is more hematologically and nutritionally balanced.Average CEA levels for pancreatic cancer were higher in the diabetic group (39.23 ng/mL vs. 12.95 in the control group); in the colon cancer group, diabetic patients had a significantly lower rate of CEA, compared with the control group (44.63 ng/mL vs. 288.85 ng/mL—p = 0.003). Lactic dehydrogenase levels were not significantly increased in comparison with the group without diabetes (308 U/L in diabetic patients vs. 330 U/L in non-diabetic subjects). The average albumin levels for diabetic patients with lung cancer were 28.5 g/L and in the non-diabetic group they were 34.9 g/L (p < 0.001). C-reactive protein levels were also significantly elevated in patients from subgroup 1  (p = 0.0022). The glycated hemoglobin level was between 5.7% and 13.1%, with an average value of 12%.In 81 patients with diabetes, the average value of hemoglobin at lung cancer diagnosis was 11.5 g/dL, versus 12.5 g/dL for the group without diabetes, and patients without diabetes (p = 0.01). Lactic dehydrogenase levels were elevated in the study group when cancer was diagnosed  compared with the group without diabetes (273 mg/dL) (p = 0.0017).In subgroup 1, the average value of hemoglobin was 11.2 g/dL, compared with the non-diabetic group’s average of 12.1 g/dL (p = 0.00023). C-reactive protein levels were also increased in the study group vs. the group without diabetes . Carcinoembryonic antigen levels, studied in pancreatic and colon cancer patients, were significantly elevated in non-diabetic colon cancer cases, with an average value of 288 ng/mL vs. 44.3 ng/mL in diabetic patients.A major difference was observed for albumin levels, showing that the nutritional balance for colon cancer in association with type two diabetes is fragile  (p = 0.006). The average LDH level was 344 mg/dL in the diabetes subgroup, and 305 mg/dL in the non-diabetes subgroup, indicating a worse prognosis of the disease in diabetic patients.Anemia at diagnosis was more common in pancreatic cancer cases with type two diabetes as comorbidity, with an average value of hemoglobin of 11.3 g/dL, against 12.4 g/dL for non-diabetic pancreatic cancer cases , against 12.9 mg/dL in non-diabetic patients. Inflammatory markers were higher in diabetes patients .p = 0.00091).There was no significant difference in albumin values . Lactic dehydrogenase levels were elevated in the diabetes group, 312 mg/dL, versus 223 mg/dL (p = 0.00029), which suggests an increased inflammation in diabetes patients, confirmed by CRP values . In the breast cancer subgroup, the average value of hemoglobin was lower in the group of diabetes patients (11.05 g/dL and 12.6 g/dL in non-diabetic patients) (p < 0.0001). The average value of HbA1c was 8.49%.Denutrition was more pronounced in patients in the diabetes group  in obese and/or diabetic patients, and to study correlations between these markers and the prognosis of cancer patients with associated type two diabetes at the onset of treatment.p = 0.002). These biomarkers may be an indicator of a patient’s inflammatory state for diabetic patients, and a neoplasm can be sought.In our study, it was found that anemia, hypoalbuminemia, elevated lactate dehydrogenase, glycated hemoglobin, and C-reactive protein levels are more pronounced in subjects with type two diabetes and cancer  and is expected to increase further, as it is estimated to rise to more than 700 million by 2045 ,18.Given the increasing interest of the European community and the Romanian government in implementing comprehensive prevention programs against the spread of cancer and type two diabetes , and theLactic dehydrogenase levels in the diabetic group for each type of cancer was studied, but the most significant difference was in the colorectal cancer (411 mg/dL vs. 273 mg/dL in the group without diabetes) and breast cancer subgroups (312 mg/dL vs. 223 mg/dL in non-diabetic cases). Increased lactate alters the microenvironment, provides nutrients to cancer cells, and leads to acidosis, inflammation, angiogenesis, immunosuppression, and radiation resistance . BonucceA major reason for the development of cancer is that the immune system loses its ability to effectively eliminate aberrant cells. High levels of lactate have a deleterious effect on immune cells infiltrating the tumor . FinallyMalignancies can also attack the bone marrow (bone marrow fibrosis), which can also lead to anemia. The bone marrow has a rich blood supply and is, therefore, a frequent site for the development of metastases . Breast The increase in proinflammatory cytokines plays a major role in insulin resistance and leads to the occurrence of micro- and macrovascular diabetic complications. Increasing IL -6 results in an anti-erythropoietic effect, as this cytokine alters the sensitivity of precursor cells to erythropoietin and promotes cellular death of immature erythrocytes, leading to a further decrease in the number of circulating erythrocytes and a decrease in circulating hemoglobin . Our stuGenetic syndromes, inflammatory bowel diseases, a history of abdominal radiation therapy, dietary factors , and tobacco use are the most incriminated factors for developing digestive tube malignancies ,28. TherThere was no difference in survival between patients with colorectal cancer who had diabetes and those who did not, according to a recent case-control study on the relationship between type two diabetes and colorectal cancer . The samFor pancreatic cancer, there is increasing evidence that inflammation plays an important role in its development . Our stuDenutrition is a known major problem both for diabetics and cancer patients. This study revealed that low plasmatic albumin levels were found in diabetic patients , compared with non-diabetic patients (35.3 g/L).Uncontrolled type two diabetes in cancer patients was also specific in our study, confirmed by an average value of HbA1c of 8.57%.Several studies suggest an important association between the depth of anemia and HbA1c levels in patients with diabetes, supporting the hypothesis that anemia is more common in poorly controlled diabetics ,33. In oCell proliferation and tissue damage are induced by chronic inflammation . DiabeteThe study limitations are represented by the limited biological tests available, with few options of state-settled biomarkers for the basic biological assessment of the hospitalized patient which can be used for this purpose.This study was not conducted by clinical trials, and its results from current practice, which show a statistically significant correlation between inflammatory biomarkers in the presence of cancer and type two diabetes, can be applied in clinical practice and can lead to improved oncological patient prognosis. Hemoglobin, LDH, CRP, and albumin levels could be used as predictive factors for cancer patients with associated type two diabetes, highlighting the importance and impact of metabolic and inflammatory disorders encountered in these chronic diseases. Anemia was strongly correlated with type two diabetes and lung, colon, breast, and pancreatic cancers; there was a higher incidence of anemia among poorly controlled diabetics. Lactate dehydrogenase levels in the diabetic patient group were significantly elevated in type two diabetes associated with cancer, versus the non-diabetic patient group. Inflammation biomarkers, hemoglobin values, and serum albumin levels are also strongly correlated, suggesting a more sustained inflammatory response, as the nutritional and biologic status is more balanced. Better diabetes control can contribute to a favorable prognosis of malignancies. The presence of diabetes negatively influences the evolution and prognosis of cancer patients, median treatment period, and survival rate.


A global survey indicates that genetic syndromes affect approximately 8% of the population, but most genetic diagnoses can only be performed after babies are born. Abnormal facial characteristics have been identified in various genetic diseases; however, current facial identification technologies cannot be applied to prenatal diagnosis. We developed Pgds-ResNet, a fully automated prenatal screening algorithm based on deep neural networks, to detect high-risk fetuses affected by a variety of genetic diseases. In screening for Trisomy 21, Trisomy 18, Trisomy 13, and rare genetic diseases, Pgds-ResNet achieved sensitivities of 0.83, 0.92, 0.75, and 0.96, and specificities of 0.94, 0.93, 0.95, and 0.92, respectively. As shown in heatmaps, the abnormalities detected by Pgds-ResNet are consistent with clinical reports. In a comparative experiment, the performance of Pgds-ResNet is comparable to that of experienced sonographers. This fetal genetic screening technology offers an opportunity for early risk assessment and presents a non-invasive, affordable, and complementary method to identify high-risk fetuses affected by genetic diseases. Additionally, it has the capability to screen for certain rare genetic conditions, thereby enhancing the clinic’s detection rate. Genetic diseases, which make up around 80% of rare diseases, are caused by variations in the genome. They can result in disabilities, deformities, and intellectual disabilities in patients, and in severe cases, they can even lead to the death of children. According to a global survey, genetic syndromes affect approximately 8% of the population . More thPrenatal screening has been used to assess the risk of a fetus affected with genetic diseases since the 1970s, with the initial focus on Trisomy 21 . MaternaUltrasonography is a low-cost, real-time, non-invasive technique for malformation diagnosis and is widely used in prenatal screening . With thThere has been significant progress in the development of deep-learning-based artificial intelligence (AI) algorithms for aiding in prenatal diagnosis ,10, espeIn this research, we developed Pgds-ResNet, a fully automated prenatal screening algorithm based on deep neural networks, to detect a variety of genetic diseases, especially some rare genetic diseases. The Grad-CAM visualization technique  was thenn = 333), from the initial list of 1000 cases, those without genetic results, 3D volume images, and complete clinical data. Thus, our dataset contained 556 normal pregnancies and 111 pregnancies with genetic abnormalities. This is a retrospective study with genetic test results available in all cases, and the genetic results were used as the gold standard for diagnosis. The decision to induce labor or not is up to the patient.From March 2020 to October 2021, the Guangzhou Women and Children’s Medical Center in China enrolled a total of 1000 pregnant women aged 23 to 38 years who underwent prenatal diagnosis . All ultThe static 3D volume acquisition of the fetal face was performed with a high-frequency probe (6–12 MHz) equipped by a GE Voluson 730 Expert/E10 . The volumes in midsagittal view were acquired using high or max quality mode and the lowest angle sweep, which allowed the inclusion of the total facial surface from the forehead to the chin. Following the collection of the ultrasound images, the sound beam angle was tilted as much as possible to 45°, the scanning volume angle range was adjusted to 50–70°, and the high-3 to max quality mode was used to set a three-dimensional volume acquisition frame of appropriate size.All fetal facial volume images obtained were analyzed offline, using 4D View software  and activating the Volume Contrast Imaging function to optimize the volume contrast resolution. This study utilized selected 3D ultrasound volume data, ensuring that the fetal head occupied 3/4 of the screen image by adjusting the magnification. Subsequently, the image was rotated by 7° and 14° to capture the fetal facial information, with the sagittal position serving as the initial plane of interception. An automated preprocessing workflow was used to remove the identification of the information and eliminate unintended human labels. Each ultrasonic image was cropped and masked to remove text, electrocardiogram and respirometer information, and other information outside the scanning sector.Overall, our dataset contained 845 normal images and 275 genetic disease images (including 12 genetic diseases) from 1000 pregnancies , with geThis study also employed data augmentation techniques to enhance the feature representation of images and prevent overfitting . Our expSpecific screening models were obtained using the fetal ultrasound images. The ResNet-18, ResNet-34, VGG-19, and VGG-16 models were trained to choose the appropriate network architecture. They are widely used in medical image analysis, especially for facial recognition ,24. ResNThe ResNet network model was obtained with 18 and 34 layers and the VGG model with 16 and 19 layers as the deep neural network model of this experiment. Initially, the training set was used following data enhancement to train the four aforementioned models, and the initial learning rate was set to 0.0001. Because there have been no relevant studies or models available for transfer learning or parameter initialization before, this study selected the random initialization method. The convolution kernel was set to , and Adam was employed as an optimizer. Subsequently, ReLU was selected as the activation function, and we applied binary cross-entropy to the output of the last dense layer, and the batch size was set to 32. The batch normalization module was added to each layer of the model, and weight decay (L2 regularization) was applied by us to avoid model overfitting. Subsequently, the epoch was set to 25, 50, 100, 150, and 200, and 5 models for each network structure were saved. The model with the smallest verification set loss was selected as the optimal model of the network structure.All the functionality, experiments, and analyses were implemented using Python  and Google TensorFlow (for the implementation of the deep learning architecture).The original patient data were divided into a training set and a test set in the experiments. TensorFlow, Keras, and Python were used for statistical analysis. The performance of the genetic diseases screening model was evaluated by calculating the area under the receiver operating characteristic curve (AUROC), sensitivity, specificity, and F1*-score on the test set. The 95% CIs were the Wald CIs for sensitivity, specificity, and AUROCs, which were calculated with empirical bootstrap containing 1000 replicates. In addition, by analyzing the output results of the model on the training set, the highest F1-value was used as the selection criterion for the best threshold value.Furthermore, receiver operating characteristic curves (ROCs) were plotted to demonstrate the screening performances and the identification of genetic diseases using the Pgds-ResNet models. The ROCs were drawn by plotting the sensitivity against the 1–specificity at different operating thresholds. Additionally, ROC analysis was performed to determine the optimal operating thresholds by using the outputs of the models on the tuning dataset. To further evaluate the performance of the deep learning model, we also used ANOVA to record the significant difference between the performance of the deep learning model and the gold standard.The Grad-CAM visual interpretation technique was employed to generate a heatmap. This heatmap highlights the areas within the images that have a significant impact on the final classification judgment, thereby enhancing the interpretability of the model algorithm. By heatmap visualization, we were able to observe the contribution of different areas of the image to the final prediction and establish potential associations between facial features and genetic diseases.The facial features of various genetic diseases are not clear. In order to make the prediction effect of Pgds-ResNet more convincing, the heatmap was compared with the ultrasound image.Three levels  of seniority sonographers participated in a comparison study. Initially, Pgds-ResNet and each sonographer received 100 images from the test set at random and determined whether the fetus exhibited certain genetic abnormalities independently. Secondly, the statistical analysis compared the diagnostic results with the genetic counseling report. We gave instructions to the sonographers that the examination time for each image was within 10 min. The sonographer was allowed to take a break for every 20 images examined.The accuracy, sensitivity, and specificity were used to compare Pgds-ResNet’s screening performance with those of the human doctors. Apart from the images, the doctors were masked from all information provided.The overview of the acquisition and pretraining processing of datasets workflow is shown in The overall framework of this research is shown in Pgds-ResNet yielded the following values on the test dataset after training: 0.98, 0.89, 0.96, and 0.92, corresponding to AUROC, sensitivity, specificity, and F1, respectively . When thp-value and F-value presented in p-value is 0.658, indicating no significant difference in performance compared to the gold standard. The F-value is 0.196, suggesting a relatively small difference. In general, the results indicate that the predicted outcomes of the models are fairly close to the gold standard, suggesting good predictive performance of the models. On the other hand, the corresponding F-values for ResNet-34, VGG-16, and VGG-19 are 15.921, 30.014, and 28.125, respectively, indicating a larger difference between the performance of these algorithms and the gold standard.The This study conducted separate statistical analyses for each type of genetic disease. The sensitivity, specificity, and F1-score of each type of genetic disease were obtained by defining the target category (a certain type of genetic disease) as positive and the other categories as negative. In screening for Trisomy 21, Trisomy 18, and Trisomy 13, Pgds-ResNet achieved sensitivities of 0.83, 0.92, and 0.75 and specificities of 0.94, 0.93, and 0.95, respectively. With the classified threshold at 0.16, the percentage of correctly classified images into positive cases was 0.83 (10/12) in Trisomy 21, 0.92 (12/13) in Trisomy 18, and 0.75 (12/16) in Trisomy 13. In total, two Trisomy 21 images, four Trisomy 13, and one Trisomy 18 were misclassified by Pgds-ResNet as normal fetuses . The misclassification is primarily due to the poor image quality of those samples , and theDuring the image classification, we found Pgds-ResNet focused on abnormalities within the facial region. The most prominent areas among 86 images of genetic diseases in our test set were the areas of the jaw, flat frontal bone, and nasal bone.The ultrasound images were used to demonstrate the screening performance of Pgds-ResNet . The conPgds-ResNet also detects rare genetic diseases and achieves a surprisingly high degree of accuracy, especially in cases without apparent fetal structural abnormalities. Pgds-ResNet detected 43 of the 45 other genetic disease images in the test set. The sensitivity and F1-value were 0.96 and 0.87, respectively . The heaWe also compared Pdgs-ResNet’s performance with three sonographers . The accTo develop a deep learning model for predicting the brain age of preterm neonates using routine clinical brain MR images, we enrolled 281 preterm infants aged 28 to 37 weeks (We propose a deep learning model called Pgds-ResNet for detecting high-risk fetuses affected by genetic diseases, especially some rare genetic diseases. Pdgs-ResNet analyzes ultrasound images and makes decisions based on the imaging characteristics that are associated with genetic diseases. Pdgs-ResNet discovers fetal facial abnormalities as the most effective features in detecting genetic diseases. The screening accuracy is on par with the senior sonographers who received genetics training before this study. Comparing the heatmaps to the ultrasound images also confirms that Pgds-ResNet is able to correctly identify Trisomy 21, 18, and 13 syndromes and a number of rare types of genetic diseases prenatally.10q25.3, 8q24, VAX1, IRF6, and other genes are associated with cleft lip disease and affect the development of the human jaw and maxilla, resulting in abnormalities in the nasal wing, cheek, lips, and other parts of the face. Rs287104 locus in the KCTD15 gene is related to the morphology of the nasal tip and alar; Rs9995821 locus in the DCHS2 gene is related to nostril aperture; Rs2977562 locus in the 3q21.3 gene is associated with the thickness of the upper lip; and Rs10176525 locus in the 2q36.1 gene is related to the height of the nasal bridge [Recent genetic studies have shown that facial abnormalities in patients with genetic diseases are closely related to the mutation of certain genes ,27. For l bridge ,31.In fact, craniofacial manifestation has aided in the screening for genetic diseases . Recent However, these genetic disease screening and diagnosis methods cannot be applied to prenatal diagnosis because the set of key facial points commonly used in facial recognition algorithms cannot be obtained in fetal ultrasound images. Our research addressed this limitation and revealed an association between fetal facial features and various genetic diseases, especially in rare genetic diseases, which enables automated screening and identification of genetic diseases based on fetal ultrasound images.The “fetal profile” plane is a required component for standard examination during pregnancy . MicrognTo better interpret Pgds-ResNet’s results and minimize the black-box effect of deep learning models, we used Grad-CAM’s visualization technique to highlight the identified abnormal regions. Pgds-ResNet discovers abnormalities primarily in the areas of the facial forehead, nasal part, mouth, and jaw in different types of cases. Comparative studies on humans and AI further showed that Pgds-ResNet could discover features imperceptible to operators. Moreover, Trisomy 21, 18, and 13; Turner syndrome; 1q21.1 microdeletion; 15q11–q13 duplication; Helsmoortel–Van der Aa syndrome; 15q26.1–q26.3 deletion; and 20p13 duplication all show distinctive variations in the heatmap.Being the first study that applies AI techniques to detect facial features in fetal ultrasound images for prenatal screening of genetic diseases, there is certainly ample room for improvement. Firstly, the limited availability of samples of fetal genetic diseases resulted in a relatively small sample size for this study. Moreover, due to the small dataset used for algorithm validation and the absence of external test sets, the results may be overly optimistic . SecondlDespite these limitations, further development of our method could make a profound impact on prenatal genetic disease screening. It is well known that training a qualified sonographer is costly and time-consuming. Rapid detection and accurate diagnosis also largely depend on a clinician’s experience. A well-verified AI model with good robustness is expected to relieve the shortage of qualified sonographers, a challenge especially in under-developed regions, and hopefully reduce the financial burdens of the patients. It will also assist clinicians in making prenatal care decisions and thus improve early intervention outcomes. It can further be used as a triage scheme in clinical practice to reduce the application of NIPT or invasive procedures and save social resources. This type of screening tool could be extremely useful and is currently not available to clinicians by any other means.In summary, this study has successfully developed an AI framework that utilizes fetal faces from ultrasound images for effective and automated screening of genetic diseases. Additionally, the framework provides informative heat maps in the context of fetal genetic conditions. Pgds-ResNet found that the fetal nose, jaw, forehead, etc., contained diagnostic information. It could help with prenatal ultrasound diagnosis, reduce false-negative results, and compensate for the lack of medical resources. However, it is worth noting that this deep-learning-based algorithm serves as an aid to doctors in diagnosis, saving them time rather than replacing them.