Different fertility preserving strategies, including ovarian tissue cryopreservation (OTC) and oocyte cryopreservation has been considered, and healthy babies have been born after successful OTC and transplantation. We evaluated follicle number and follicle health in ovarian tissue from a cohort of BT and SCD patients who underwent OTC before the age of 18 years. Patients undergoing OTC from 2002 to 2019 were included. A total of 14 girls and adolescents with BT and four with SCD, aged 2.8–17.4 years at OTC were included together with a reference group of 43 girls and adolescents with non-anemia diseases considered to have normal ovaries aged 0.6–17.9 years at OTC. Ovarian follicle density was measured in cortex biopsies and compared to the reference group. Expression of proteins associated with follicular health was evaluated using immunohistochemistry. Follicles were detected in the ovarian cortex biopsies from all patients with BT and SCD. The follicle densities were within the 95% prediction interval of the reference group in all cases. A similar expression of six proteins essential for follicular health was detected using immunohistochemistry in BT, SCD, and references. OTC should be considered an option for young girls and adolescents with BT and SCD.Women with  Arouβ-thalassemia (BT) is a hemoglobin disorder caused by a deficiency in one or more β-globin genes causing abnormal hemoglobin formation and thereby reduced oxygen caring capacity of the erythrocytes  and SCD may lead to iron overload, resulting in liver damage, cardiac complications, endocrine dysfunctions, and compromised function of the reproductive organs  derived from a HLA-compatible bone marrow donor can be curative in young BT and SCD patients , 24–26. The aim of the present study was to evaluate the number of ovarian follicles, the follicle morphology, and the protein expression of essential follicle and oocyte health markers in girls and adolescents with BT and SCD undergoing OTC.A total of 14 girls and adolescents with BT and four with SCD aged 2.8–17.4 years (mean age ± SD 10.4 years ± 4.2) had one entire ovary removed by laparoscopy for fertility preservation by OTC before HSC. Clinical data is listed in The ovarian cortex was isolated and cut into small pieces as previously described for slow-freezing –34 and sTissues were fixed in Bouin’s solution and embedded in paraffin. Tissues for density estimation were cut in 15 to 30 µm serial sections, de-paraffinated in xylene and stained with periodic-acid Schiff and counterstained with Mayer´s hematoxylin and erosin reagents (Sigma-Aldrich) for assessment of cell morphology. Sections of 5 µm were processed for immunohistochemical (IHC) staining.3). In Copenhagen, the follicle density was estimated in 15 to 30 µm section using a mathematical model described by Schmidt and colleagues (α) to account for the possibility of counting the same follicle more than once. Since the mean diameter of a primordial follicle is 44 µm , and incubated with secondary horseradish peroxidase (HRP) conjugated antibody  for 30 min at room temperature, visualized with DAB detection system , and counterstained with Meyer’s hematoxylin . Details of antibodies and conditions are given in Ovarian tissue sections were de-paraffinated, rehydrated in ethanol followed by antigen retrieval in either 10 mM sodium citrate, pH 6 or 10 mM Tris, pH 9. Endogenous activity was inhibited using 1.5% peroxidase, nonspecific binding was inhibited using 1% bovine serum albumin (BSA) . Sections were incubated with primary antibodies overnight at 4°C, washed in phosphate-buffered saline with Tween20The GraphPad Prism 8.0.0 program  was used for statistical analysis. Linear regression was used to evaluate follicle density against age. Mann–Whitney U-test was used to evaluate if the BT and SCD follicle densities were different from the reference group. Statistical significance was defined as p-values <0.05.In all cortical biopsies from BT and SCD the follicles appeared morphologically normal. Most of the primordial follicles presented with a centrally located primordial nucleus surrounded by a single layer of orderly flattened granulosa cells. Atretic follicles and abnormal follicles with irregular granulosa cell layers were also detected, which is common in children ovaries , 41; exaImmunohistochemical analysis (IHC) was used to detect the presence of two oocyte specific markers growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) –44, and Two patients with BT (subjects 6 and 7), both aged 9 years at OTC had five ovarian cortex pieces  transplanted in Denmark at the age of 22 and 23 years . SubjectThe present study characterized ovarian follicles in girls and adolescents with BT and SCD and explored the feasibility of fertility preservation by OTC in these patients. Follicle density, morphology, and the expression of essential follicle- and oocyte specific proteins in early stage follicles were comparable to an age-matched reference group. In all cases the estimated numbers of primordial ovarian follicles were within the 95% prediction interval of normal age-matched reference ovaries, illustrating that the number of follicles is not compromised by these diseases during childhood and puberty. BT and SCD patients originated from Denmark , Australia , and UK (1 BT), whereas all reference ovaries were Danish. It cannot be excluded that the evaluated parameters would have been different in an Australian/UK reference cohort, although the methods used were cross-validated and no differences were found . In all In four cases, there were ovarian material available for IHC analyses, and the expression of three proteins involved in the regulation of folliculogenesis   and oocyTaken together, these findings suggest that the pool of primordial follicles, is normal. This argue in favor of performing OTC, which preserves the primordial follicles only. OTC is indeed indicated before HSC treatment which carries a significant risk of subsequent infertility. When the tissue is transplanted after HSC the iron level and the endocrine milieu will be normal and normal follicular growth will be expected. OTC may also be considered in girls who are not facing HSC transplantation, since it is not clarified whether a prolonged systemic high iron level will compromise follicle health on the long run.Two patients in the cohort had tissue transplanted (subjects 6 and 7). Both cases had tissue frozen before puberty at the age of 9 years. These patients were 22 and 23 years of age and menopausal at transplantation; in both cases serum hormone levels went back to normal three to four months after transplantation, illustrating the functionality of the cryopreserved pre-pubertal ovarian tissue. Subject 7 became pregnant following IVF treatment within a year from transplantation and gave birth to a healthy boy, being the first proof of concept of truly pre-pubertal ovarian tissue giving rise to healthy offspring .Globally, successful OTCs have been reported in three BT cases and six SCD cases , 52–58. The present study found no differences in the density of primordial follicles, morphology, and expression of follicle- and oocyte specific proteins in girls with BT and SCD compared to the age-matched reference group, suggesting that primordial follicles may not be affected by iron overload. These findings together with the reported cases of successful ovarian tissue transplantations giving rise to life-births in women with BT and SCD, suggest that OTC should be offered to girls and adolescent with BT and SCD, before HSC. OTC may also be considered in young BT and SCD patients, who are not facing and HSC transplantation since it is not clarified whether a prolonged systemic high iron level will compromise follicle health and fertility in the long run.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher on request.This study was approved by the Ministry of Health (J. no. 30-1372) and by the Danish authorities to comply with the European Union tissue directive. Written informed consent to participate was provided by the parrents /participants’ legal guardian.LM designed the project, wrote the paper, cryopreserved ovarian tissue, measured follicle density in the Danish cohort, did IHC staining, and analyzed the data. CA, SK, SP, and DG cryopreserved ovarian tissue. DG measured the follicle density in the Australian cohort. JB assisted in statistical analysis. EE and KM recruited patients and did the ovariectomies. AK and BK designed antibodies (GDF9 and BMP15), contributed to design and interpretation of the data. CA designed the project and wrote the paper. All authors contributed to the article and approved the submitted version.The study was designed, conducted, analyzed, and reported entirely by the authors. This paper presents independent research funded by grants from Rigshospitalets Forskningspuljer, the International EU project ReproUnion.AK and BK were employed by the company Ansh Labs.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Overall, the study enumerates the development of an automated pencil stroke device for fabricating graphite electrodes, which can potentially be harnessed in numerous miniaturized paper based applications.Graphite pencil stroked electrodes for paper-based Microfluidic devices are gaining immense attention due to their electrochemical properties, cost efficiency, and ease-of-use. However, their widespread use has been hindered by the challenges associated with their manual fabrication such as non-uniformity in graphite deposition, applied pressure, etc. This work presents the design and development of an automated graphite pencil stroking device for graphite electrode fabrication with high efficiency through a compact, inexpensive and automatic process, with reduced fabrication time and human intervention leading to more uniformity. The motion platform of Graphtec plotter was used to create multiple strokes with the help of the proposed device. Such inexpensive graphite electrodes (less than the US $1) have been observed to be porous in nature, acting as diffusion agents. The automated graphite electrodes were used to study the performance of microfluidic paper fuel cells (MPFCs) with formic acid, oxygen, and sulphuric acid acting as fuel, oxidising agent and electrolyte respectively. From this configuration, the maximum current density and power density were measured to be 1,305.5 µA cm Paper–pencil based microfluidic fuel cell is one of the most recent vital advancements to develop point of the source (POS) and point of care (POC) devices owing to the well-proven benefits of microfluidic environment and graphite electrodes5. In recent years, graphite pencils are being used as electrodes and have shown encouraging outcomes and promising features of MPFCs when compared with the existing approaches. Initially approaches on graphite electrodes, Aoki et al. demonstrated the first use for of graphite material (pencil lead), as electrodes6. Bandapati et al. investigated the performance of membraneless glucose biofuel cell with various grades of graphite pencils act as electrodes8. Rewatkar et al. examined 3D printed enzymatic biofuel cell performance with graphite pencil as an electrodes9.Microfluidic devices with integrated electrodes, or electromicrofluidic devices, have attained essential roles in diverse areas, including energy harvesting for portable applications and sensing devices11. Veerubhotla and Ye et al. studied performance with graphite pencil stroke electrodes on paper based microbial fuel cells13. Lal et al. showed a paper based microfluidic fuel cell using different filter papers, whereby graphite sheets acted an electrode, and formic acid and potassium permanganate acted as a fuel and an oxidant, respectively14. Shen et al. studied the influence of performance on paper based fuel cells at fluidic concentration variation15. Further, the required electrodes can be integrated into the microchannel with sufficient distance between electrodes, from the depletion to the diffusion zones, to reduce the effect of fuel mixing16. In the earlier work by the authors, Rao et al. investigated pencil paper based fuel cells with formic acid acting as fuel, oxygen as an oxidant, graphite electrodes as electrodes. Such graphite electrodes were realized by manual strokes with varying numbers of strokes and with diverse grades of pencils18. Jung et al. investigated and presented a paper based vanadium co laminar micro fuel cell where electrodes were fabricated by screen printing of water dispersible graphene paste. They reported a maximum current density of 550.5 μA cm−2 and 445.0 μA cm−2 for graphene paste electrodes and manual pencil stroked electrodes fuel cell, respectively19. Overall, in most of the reported work, the pencil electrodes have been physically realized.In spite of the numerable benefits of such devices, they come with an inherent setback of the physically fabricated electrodes with non-uniform deposition of graphite due to the subjective nature of the applied pressure and manual counting of the number of strokes. Most of the recent researches in the field of paper–pencil based microfuel cells employ manual deposition of graphite on the paper for fabricating the electrode. Arun et al. reported formic acid microfluidic fuel cell with manual graphite pencil electrodes with diverse grades of pencils18. This manual process for electrode fabrication brings in scope for many precisions related variations and errors due to human intervention, as the designer does not have any precise control over the amount of graphite deposited. Further, the amount and quality of the graphite being deposited and adhered on the surface can vary from person to person and time to time depending upon the pressure applied. The quantity of graphite deposited on the paper affects the performance of the electromicrofluidic device and can be modified by varying the number of pencil strokes. Based on this, it can be concluded that there is a need for a simple, user-friendly, and reliable platform device for graphite electrodes formation in an automated manner.Evidently, the quantity of graphite deposited on the paper affects the performance of the fuel cell, which can be modified by varying the number of pencil strokesThe present work describes the design and development of an automated graphite pencil stroking device for graphite electrode fabrication with uniformity in applied pressure during electrode fabrication leading to even graphite deposition on porous cellulose paper. The proposed device addresses the aforementioned problems and provides an integrated solution for economical, easy to use, uniform and, a portable, automated device for graphite electrode on for paper based devices. Such automated graphite electrodes have excellent uniformity, high efficiency, and can be fabricated inexpensively. Moreover, the quantity of force can also be identified at the time of graphite electrode fabrication time by force sensing resistor (FSR) sensor. The platform has been harnessed to be used to realize microchannel and electrodes of microfluidic paper fuel cell (MPFC) showing excellent power output. Overall, the platform is an accurate, fully-automated device for graphite electrode fabrication on porous cellulose paper for sensing, energy harvesting, and flexible electronics applications.2. Here, two diverse grades of graphite pencils, HB and 8B, were used to prepare electrodes with various strokes, with constant force and uniform graphite particle deposition. The choice of the graphite pencils was based on the earlier published work by our group18.Graphite pencil electrodes were fabricated on porous natured Whatman filter paper Gr 1) by automated pencil stroke device with Graphtec plotter for microfluidic paper fuel cells (MPFCs). As shown in Fig.  by automAs shown in Fig. Subsequently, the force was identified by the FSR sensor at the time of the fabrication of the graphite electrodes. The step-by-step force-sensing operation of the FSR sensor is mentioned in Fig. S1. The value of force for various graphite pencil strokes with an error bar is shown in Fig. The surface morphology of automated and manual graphite pencil stroke electrodes, fabricated on porous nature Gr1 Whatman cellulose filter paper with graphite pencils , at 50 strokes, were captured by scanning electron microscopy (SEM) and compared. As can be seen in Fig. In Fig. Further, surface elemental characterization was performed by EDX analysis (energy dispersive X-ray). The difference between automated and manual composed graphite electrodes was observed, and surface elemental composition was evaluated with standard deviation. As shown in Table 21. The force (N) was measured at the time of electrodes fabrication, and an explanation is given in “18. The complete experimentation setup with various automated graphite electrodes  is shown in Fig. S2.After successful graphite electrode fabrication using the proposed platform, such electrodes were utilized in a microfluidic paper fuel cell (MPFCs) with formic acid (1 M) acting as fuel, sulphuric acid 3.75 M) as an electrolyte, and oxygen as oxidant. Here, two different automated graphite electrodes  were prepared with 30, 50, and 100 strokes with equal force and uniformity. From the literature, the graphite content present in graphite pencils were 68% in HB and 91% in 8B5 M as anInitially, an Open Circuit Voltage (OCP) was observed in a MPFC with automated graphite electrodes. Subsequently, the polarization performance of MPFC was investigated by using the chronoamperometry technique with stable OCP of 300 mV. The microfluidic fuel cell polarization curves with various automated pencil strokes  are shown in Fig. 2 and 135.504 µW/cm2, respectively, with 100 automated pencil strokes.The electrochemical characterization of MPFCs was carried out with several automated pencil electrodes with a varying number of strokes. These characterizations were carried out based on energy density  with the measured open circuit potential (V). In such configuration, the maximum current and power densities were observed to be 1,305.6 µA/cmThe aforementioned automated graphite pencil stroke device with plotter has shown excellent compatibility, robustness, and reproducibility. The fabrication process of the uniform electrodes is simple and inexpensive compared to the manual graphite stroke electrodes. It is clear that automated graphite pencil stroke device has a strong potential to enable various low-power applications, flexible electronic application, and sensing applications.The device has a promising potential for large scale fabrication of automated electrodes with uniform deposition and constant force at the time of electrode fabrication. Such fabricated electrodes can be utilized for energy harvesting, flexible electronics, and sensing applications.Different grades of graphite pencils such as HB, 8B, and a varying number of strokes can be used to obtain electrodes with varying properties and efficiency on porous Whatman filter paper as per the requirement with the help of automated pencil stroke device with graphtec plotter.2 and 135.504 µW/cm2 respectively at 0.3 V stable OCP at 100 automated pencil strokes.After rigorous experimentation, characterization, and analysis, MPFC with automated graphite pencil electrodes show the optimum performance with power and current densities of 1,305.6 µA/cmThe proposed device also ensures uniform application of force at the time of electrode fabrication. The fabricated automated electrode device is very inexpensive (less than the US $1) and electrodes fabricated with this device can be used in a wide variety of applications.The proposed device provides a platform to develop standardized electrodes, which can be used for various applications where the electrical output is needed. These applications include microfluidic fuel cells, electrochemical and amperometric sensors for toxic and nontoxic biochemical etc.Standardized electrodes on paper substrates can be further used to carry out various processes of electrically sensitive organic molecules and analytes in miniaturized settings.The utility of the proposed device can also be extended to develop standardized electrodes with wax, correction pens or other similar materials for diverse point-of-care applications.This paper presents the design and development of an automated pencil stroke formation device for electrode fabrication. Following are the main conclusions and the prominent features of the proposed device:All chemicals were purchased from Alfa Aesar, India. Different pore sizes of Whatman filter paper Grades such as Gr 1, 6 were purchased from Sigma Aldrich, India. Multiple grades  of Graphite pencils (Apsara brands) were purchased from a local stationery store. Graphite pencils  were used for electrodes fabrication by automated pencil stroke device with uniformity, and constant force on porous nature filter paper. A PLA (polylactic acid) filament  was used to fabricate the automatic pencil stroke device and its components by FDM based dual extruder 3D printer . The Force Sensing Resistor (FSR) sensor was used to identify the force (N) at the time of graphite electrode fabrication. The FSR sensor  was procured from Interlink Electronics, India. Graphtec CE 6000-60 cutting plotter (X–Y motion) was used to fabricate graphite electrodes. Graphtec CE6000-60 cutting plotter was purchased from Graphtec America Inc. CE 6000 series, US.22.Prior to the automated pencil stroke device fabrication, FDM based 3D printer was used to fabricate an automated pencil stroke device and its components with selected dimensions. In the beginning, virtual design of an automated pencil stroke device, with various components, was created with the help of SolidWorks 2013 modeling software. Detailed step-by-step fabrication procedure and prototype of an automated pencil stroke device is mentioned in Fig. As can be seen, a spiral spring, with bolt and nut mechanism, was used to adjust the force on the FSR sensor and graphite electrodes fabrication time to maintain constant force and uniformity of graphite particles deposition on the porous cellulose paper (Whatman Gr 1 filter paper).Graphtec plotter was used to hold the pencil stroke device for electrodes fabrication with varies multiple strokes and to draw the pencil strokes on a porous paper based substrate. The complete experimentation setup of an automated pencil stroke device with plotter is illustrated in Fig. The complete schematic representation of graphite pencil holder with the motions (XYZ orientations) mechanism of the plotter is depicted in Fig. Here, two technical supporting softwares were employed for drawing graphite pencil strokes: Graphtec studio (GS) for operating the Graphtec plotter and AutoCAD to design the electrodes configuration. After designing the geometry of the electrodes, it has been converted into .dxf file and imported into Graphtec studio (GS) for automated pencil strokes. The complete outline representation of graphite electrodes fabrication with graphtec plotter motions , graphite electrodes design procedure is mentioned in Fig. Evidently, most of the electrodes, on a paper microfluidic device, are fabricated manually and therefore are without uniformity in the quantity of force applied on electrodes. To overcome this, an FSR passive sensor was used for identifying the amount of force applied on electrodes at a particular fabrication time. For the FSR sensor, a microcontroller was used to operate a sensor for identifying the applied force on electrodes through MATLAB coding. This microcontroller was connected to the PC to communicate the force data. Afterwards, the Arduino Uno app was used to execute a Matlab program for capture, analyse, and tune the Force data.Supplementary Information 1.Supplementary Video S1.Supplementary Video S2.Supplementary Video S3.


We studied the stress response of five-year-old Scots pine xylem to mechanical wounding using RNA sequencing. In general, we observed a bimodal response in pine xylem after wounding. Transcripts associated with water deficit stress, defence, and cell wall modification were induced at the earliest time point of three hours; at the same time, growth-related processes were down-regulated. A second temporal wave was triggered either at the middle and/or at the late time points (one and four days). Secondary metabolism, such as stilbene and lignan biosynthesis started one day after wounding. Scots pine synthesises the stilbenes pinosylvin and its monomethyl ether both as constitutive and induced defence compounds. Stilbene biosynthesis is induced by wounding, pathogens and UV stress, but is also developmentally regulated when heartwood is formed. Comparison of wounding responses to heartwood formation shows that many induced processes (in addition to stilbene biosynthesis) are similar and relate to defence or desiccation stress, but often specific transcripts are up-regulated in the developmental and wounding induced contexts. Pine resin biosynthesis was not induced in response to wounding, at least not during the first four days. Trees handle these challenges with preformed and induced defences. For example, in Scots pine (Pinus sylvestris L.) the thick bark that contains heavily lignified cells acts as a primary barrier against harmful intruders and is a preformed passive defence3. The phloem and xylem of pine contain toxic compounds such as resin acids in resin ducts and these serve as a second layer of preformed defence, if the primary barrier fails to stop the invasion4. The pine stilbenes, pinosylvin (PS) and pinosylvin monomethyl ether (PSME), are secondary metabolites that play a dual role in both preformed and induced defence. They are induced by herbivory, wounding and pathogen attack in, e.g., xylem and needles, but form constitutively under developmental control when the sapwood passes into heartwood and is filled with extractives. Stilbenes are derived from the phenylpropanoid pathway, starting from conversion of phenylalanine to cinnamic acid by phenylalanine ammonia lyase (PAL). Although pine stilbene synthase (STS) is well characterised, the enzyme that converts cinnamic acid to cinnamoyl-CoA, the substrate for STS, is not identified in pine, and the pinosylvin O-methyltransferase (PMT2) responsible for PSME formation was only recently identified correctly5. Regulatory proteins triggering stilbene biosynthesis in Scots pine are not known at all.Angiosperm and gymnosperm trees must deal with a variety of environmental challenges, including attempts of invasion by herbivorous insects and pathogenic fungi6. They showed that concentration of stilbenes, resin acids and lignans was increased in the xylem next to the wound. Three months after wounding, PS and PSME concentration in xylem had increased from levels below detection to 3.8 and 6.7 mg/g dry weight, respectively. Other experimental treatments leading to PS and PSME accumulation, and to transcriptional induction of the STS gene in pine, include ozone fumigation7, fungal elicitation8, elicitor induction9 and ultraviolet light (UV-C) irradiation10.Harju and colleagues conducted a mechanical wounding study on stems of three years old Scots pine seedlings11. Chemical analysis of pine stems after wounding showed that, in addition to stilbenes, concentration of resin acids and lignans was highest in the area next to the wound6. Resin acids are indeed released from resin ducts in response to herbivory and mechanical wounding13. Once secreted, volatile terpenoids gradually evaporate from the wound areas while non-volatile diterpenoids polymerise and harden, eventually sealing the wound4.Resin acids (non-volatile diterpenoids) along with volatile monoterpenoids and sesquiterpenoids, are synthesised in resin duct epithelia during tree development, but are also induced in response to stress16. Lignans have been reported to play important roles in plant defence as phytoalexins17. However, they are also developmentally regulated and in Scots pine lignans can be found in knotwood18.Lignans are derived from the same monolignol subunits as lignin. They are dimers of monolignols and, unlike lignin, optically active. Stereospecific dimerization is guided by dirigent proteins (DIR) that as such do not have enzymatic activity6 measured extractives in stems of Scots pine seedlings in order to calculate genetic parameters of their induction. Half-sibs used in the experiment showed very high heritabilities (h2) for wound induced PS and lignan concentration (0.71 – 1.03). Heritability was also estimated between the wound induced extractives in the seedlings and the developmentally programmed amount of extractives in the heartwood of their adult mother trees. The relatively high offspring-parent heritability for PS of 0.31 suggests shared genetic control of developmentally regulated and induced biosynthesis of PS.Harju and colleaguesSTS), find candidate regulatory genes for the pathway and to characterise the wounding response in the young xylem (without a pathogen inoculum) in general terms. Many stress response studies have been conducted on either the bark or the phloem, or both, with a portion of xylem (sapwood) included23, but in all of these studies wounding was combined with a pathogen inoculum. Finally, we wanted to extract similarities and differences between the wound response and heartwood formation, both processes being characterised by stilbene biosynthesis in Scots pine. The high parent–offspring heritability between heartwood and wound induced stilbene biosynthesis brings up a hypothesis that there are genetic components, e.g. regulatory genes, that are shared between the wounding response and heartwood formation.We used here the same experimental setup, now with five years old seedlings, to investigate how the transcriptome of young Scots pine xylem reacts to mechanical wounding in the time scale of few hours to few days. Our aim was to define genes involved in PS and PSME biosynthesis (in addition to STS (key enzyme of pine stilbene pathway) expression of the wounded and unwounded stems (with bark and phloem removed) with semiquantitative RT-PCR  after wounding, reached a steady level at 24 h (1D), showed the highest level at 96 h (4D) and then declined. We sequenced RNA of stem samples at these time points  using the SOLiD platform. Differential expression analysis using edgeR showed that out of the total 77 326 tentative consensus sequences in the Pinus EST collection  17 584 survived the filtering for very low or missing expression and a total of 4595 transcripts were differentially expressed  in the stem (xylem) at 3H, 1D or 4D after wounding, compared to the control (0H) (Supplementary Tables Pinus EST collection was 88% (Supplementary Table Pinus EST collection is a combined collection of expressed sequence tags from different pine species and due to high sequence similarity between the species they have been assembled into ‘tentative consensus’ (TC) sequences as if they originated from a single taxon24. The nature of the Pinus EST collection was discussed in our earlier study of heartwood formation25. The main advantage in using the Pinus EST collection for mapping, instead of an assembly generated from our data, is that we can directly address the expression level of genes that are practically not expressed, such as the resin acid biosynthesis genes during heartwood formation25. For comparison, we also converted the SOLiD platform colour space reads to nucleotides, assembled them using Trinity26, and then mapped the reads to the annotated assembly. The average mapping rate to the Trinity assembly was 77%  biological function terms of up- and down-regulated transcripts at each time point were used for the analysis.The 4595 differentially expressed TCs were subjected to gene set analysis (GSA) using the R package 32 or were defence-related37  was up-regulated in response to the drilling trauma  were up-regulated in the xylem at 3H after wounding Fig. a. The couma Fig. a,b. The One and four days after wounding GSA shows a very similar pattern, which differs dramatically from the 3H time point. Only the ‘biosynthetic’ and ‘secondary metabolic’ processes were up-regulated at all three time points. ‘Translation’ that was down-regulated at 3H, was up-regulated at 1D and 4D. Similarly, at one and four days after wounding, transcripts annotated with ‘embryo development’ were up-regulated  in at least four libraries were subjected to hierarchical clustering analysis using the Bayesian hierarchical clustering algorithm SplineCluster40. Although SplineCluster distinguishes 21 clusters with the parameters used  show upregulation at 3H, which then returns to control levels during 1D and 4D. In clusters 1 and 2 (173 TCs) this pattern is very prominent. Another group of genes  reacts more slowly and peaks at 1D, returning towards control at 4D. The third distinctive pattern  is shared by genes that are constantly up-regulated (compared to the control) until 4D, the actual peak of expression not visible in the experiment. One fifth of the differentially expressed genes showed a down regulation pattern during the time course  and their expression does not recover during the time course. The two largest clusters are numbers 18 and 19 , showing a flat or slightly up-regulated pattern. In these clusters only 1.4% of the genes are differentially expressed, by the criteria we used (FDR < 0.05). Most of the other patterns are populated nearly exclusively by genes labelled differentially expressed by edgeR’s statistical analysis  enrichment analysis was carried out for differentially expressed TCs in each cluster and significantly enriched (FDR < 0.05) GO terms are listed in Supplementary Table −100, all relate to response to abiotic, biotic and endogenic stimulus, and to stress   gradually decreased in their expression at 1D and 4D after wounding. To summarise, Scots pine triggered two temporal patterns of defence after wounding: a transient one at 3H and a second wave starting at 1D after wounding.Other defence related transcripts, such as those encoding ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), EDS1-like protein, PHYTOALEXIN DEFICIENT 4 (PAD4) and SENESCENCE ASSOCIATED GENE 101 (SAG101) were triggered already at 3H after wounding. Transcripts encoding nematode resistance protein HSPRO2, elicitor-responsive protein, and the whitefly induced protein Gp91-Phox, and a pleiotropic drug resistance protein were also triggered 3H after wounding. These early-triggered defence transcripts  were induced after wounding, some of them as early as 3H after treatment. In general, transcription levels of these TFs were resorted towards the control at late time points. Several transcription factors were moderately down-regulated after wounding and grouped in cluster 1–3). Others reacted more slowly and stayed up-regulated at 4D . Still others showed moderate down regulation (cluster 16).Of the differentially expressed TFs, plant WRKY TFs stand out . Wounding of pine stem also induced transcription factors that belong to the APETALA 2/ethylene responsive factor (AP2/ERF) superfamily , as well as pinosylvin O-methyltransferase 2 (PMT2) were induced 1D after wounding. The expression level of these transcripts continued to increase at the 4D time point -α-pinene synthase, diterpene synthase and abietadienol/abietadienal oxidase CYP720B), were not correlated and showed different expression profiles in response to wounding. (-)-α-pinene synthase, responsible for biosynthesis of one of the volatile terpenes0B, were  12 Fig. , and was 15 Fig. . Diterpe25 and during wounding was done to uncover further shared gene activities in these processes. The RNA-seq libraries in our previous work on heartwood formation in Scots pine25 were mapped against the same Pinus EST collection, version 9.048, with same criteria as the libraries in this study.The pine stilbenes PS and PSME are synthesised both during heartwood formation and after wounding of stems. Strong induction of STS and PMT2 encoding transcripts can be observed in both cases, and in both cases drying of the tissue takes place. Comparison of the transcriptomes in the transition zone to heartwoodBy comparing the differentially expressed transcripts between the wounding stress response and the transition zone transcriptomes, we found that a total of 424 TCs were coregulated in these conditions  of the xylem. Strong induction of ERD, LEA, LP3 and dehydrin proteins was observed in the earliest time point (3 h). These proteins play a role in drought tolerance when the plant is under dehydration or cold stress51. A second temporal wave of defence was triggered at the middle and latest time points. These transcripts encoded antimicrobial peptides, chitinases, defensin, elicitor response proteins, pollen allergen, PR proteins, and thaumatin-like proteins. They have antimicrobial and antifungal activities and respond to elicitors and stresses caused by invasion of bacterial, insect, and fungal pathogens, or methyl jasmonate treatment and stresses such as cold55. Wounding by drilling through the stem (however without a concomitant pathogen inoculum) apparently mimicked a severe invasion of pathogens. Scots pine activated several defence pathways in order to encounter with potential invaders, as wounding exposed the inner xylem to the outside world.Besides dehydration stress-related genes, Scots pine responded to wounding with induction of genes encoding EDS1, PAD4, SAG101, elicitor-responsive and nematode resistance proteins at the earliest time point. EDS1 interacts with PAD4 and SAG101 to trigger defence against pathogen invasion and mediates the plant defence hormone salicylic acid biosynthesis57. Rapid induction of transcripts encoding these enzymes at early time points suggests that cell walls of wound areas started to undergo modification and remodelling very quickly.The cell wall modification enzymes expansin, endoglucanase, and XTH are associated with modification of the cell wall by cleaving its hemicellulose-cellulose network to provide extensibility for growth6. Dirigent and dirigent-like proteins have been associated with response to abiotic/biotic stress and were induced also in spruce, in response to wounding and white pine weevil or western spruce budworm herbivory59. Besides lignans, pine stilbenes were synthesised in the xylem next to the wound6. We also observed that pine stilbene biosynthetic transcripts were induced in xylem at 1D and 4D as a response to wounding stress. Altogether, it can be concluded that pine was remodelling the cell walls at wound areas in order to maintain cell wall integrity, prevent invasion of pathogens, enhance mechanical strength for turgor pressure and to prevent further water loss.The induction of lignan biosynthetic transcripts such as PLR, dirigent and dirigent-like protein at 1D and 4D after wounding implies that lignan deposition takes place in wounded areas. This is in accordance with our earlier study, where lignans were found accumulating in the pine xylem next to wounding12. It was somewhat surprising that transcripts involved in resin acid biosynthesis and its upstream pathways were not induced in the xylem as a response to wounding in our data. Harju and colleagues6 observed that high amount of resin acids accumulated next to the wounded areas when they carried out a chemical analysis three months after wounding. It is possible that resin acid biosynthesis starts later after wounding, or that accumulation of resin acids in the wounded areas is exclusively due to transfer of resin through resin ducts.Pine resin acids can be found in the bark, sapwood and heartwood. They act as defence barriers against herbivore invasion, particularly in the bark25. These two transcription factors, however, were not induced in response to wounding. MYB TFs, like NAC TFs, are involved in secondary cell wall formation and are common regulators of plant secondary metabolism61, often in response to biotic stresses62. MYB TFs were reported regulating poplar (Populus tremuloides) secondary metabolites in response to abiotic (wounding and UV-B irradiation) and biotic (pathogen) stresses62. NAC transcription factors have roles in secondary cell wall formation60 but they are also involved in response to insect herbivores, fungal infection, wounding, cold and heat shock and dehydration65. In the results reported here, in addition to MYB and NAC, a wide array of TFs such as AP2/ERF, AP2, bHLH, DREB, ERF, MYC and WRKY was strongly induced either at 3H or 1D after wounding. In general, their expression level was reduced at 4D. AP2/ERF transcription factors have been reported to play a role in regulating plant responses to pathogen attack, wounding, abiotic stresses like cold, salinity and drought67. Members of the large WRKY TF family have been associated with plant defence69 and have been reported to play roles in response to biotic and abiotic stresses72.One MYB and one NAC transcription factor was up-regulated during heartwood formation and was highly correlated to phenylpropanoid biosynthesis in our recent study6, comparison of the transcriptomes in the transition zone to heartwood25 and during wounding (data presented here) may uncover further shared gene activities in these processes. By comparing the differentially expressed transcripts between the wounding stress response and the transition zone transcriptomes, we found that a total of 424 TCs were coregulated in both conditions , but also its upstream pathways of phenylalanine biosynthesis (the shikimate pathway) and sugar metabolism, all the way to sucrose synthase, a general marker for metabolic activityAn obvious explanation is that these pathways are already active in the unwounded xylem of the five-year-old Scots pine seedlings, which were active in growth and lignin biosynthesis when the experiment started. Indeed, the upstream pathways for stilbene (and lignin) biosynthesis are already active in the unwounded stems and in most cases, are not up-regulated further Fig. .The exceptions to this pattern are interesting. For the entry point activity (PAL) of the phenylpropanoid pathway , the transcripts that were induced during heartwood formation are constitutively active in the wounding experiment. Interestingly, in addition, we can observe PAL transcripts not active in the transition zone but either constitutively active in the young stems or induced by mechanical wounding. A similar pattern is observed for 4CL transcripts, and for many others the gene expressed constitutively in the wounding experiment is not the one that is induced in heartwood formation Fig. .During heartwood formation, two transcription factors showed upregulation in the transition zone, a member of the MYB and the NAC family. TCs encoding these proteins were not up-regulated during wounding, instead other MYB and NAC family transcription factors were , eastern Finland. The trees were 39 years old during seed collection. Seeds from each mother-tree comprise a half-sib family , in which the fathers originate from an unknown stochastic pollen pool. The seeds were sown into plastic trays in May 2005 and were grown in a greenhouse at the Punkaharju Research Unit , Finland. During five years of growth, a sufficient level of irrigation, a low level of fertilization and no artificial illumination was applied. Aphids were controlled with pesticide . The mechanical wounding experiment was performed in May 2010 at the beginning of the sixth growing season. Ten 2.5 mm diameter holes with 1 cm interval were drilled through the stem on the fourth internode counted from the top of the seedling. Initial test sampling was carried out 0 (control), 3, 6, 12, 24, 48, 96 and 192 h after wounding. For RNA sequencing, three seedlings from each of the four families were treated and sampled 3, 24 and 96 h after wounding, respectively, and one as a control. Wounded stem sections and the corresponding section of the unwounded control were cut apart, and their bark and phloem were removed. The harvested stem sections were cut into pieces and frozen in liquid nitrogen immediately. The samples were kept at -80 °C before RNA extraction.25. For real-time quantitative RT-PCR (qPCR), cDNAs of each time point were pooled and qPCR was performed as described and with the primers described in our earlier study25. Sample preparation for RNA-seq for control and 3, 24 and 96 h after wounding was carried out as described earlier25.The frozen stem samples were further cut into smaller pieces and ground to fine powder using a milling machine  with intermittent cooling with liquid nitrogen. Total RNA isolation, genomic DNA digestion, purification, RNA quantitation, and semiquantitative STS RT-PCR with first strand cDNA that was synthesised from a total of 500 ng of total RNA as template was performed as described in our earlier studyPinus EST collection, version 9.0 obtained from Pine Gene Index48 using the SHRiMP277 alignment tool. Mapping parameters and data handling after mapping were performed as described earlier25. The computational tasks were carried out on the Finnish Grid Infrastructure clusters and supercluster server in CSC- IT Center for Science Ltd, Espoo, Finland. Table of mapped read counts for the RNA-seq libraries was imported into an edgeR R session78. The low expression transcripts of Pinus ESTs collection were filtered, and only TCs that achieved at least eight counts per million (CPM) in at least four libraries were kept. Differential expression analysis was carried out as described before25.Paired-end colour space reads of control, 3, 24, and 96 h after wounding RNA-seq libraries were mapped against the Pinus EST collection annotations are either missing or not informative. We updated the annotations as described in our previous study25. The original and updated annotations are listed in Supplementary Table Some of the Trinity software26 with default assembly parameters, except that SS_lib_type (library type parameter) and min_kmer_cov (minimum kmer threshold abundance) was set to FR and 2, respectively. The annotations of the Trinity assembly were obtained using the blastx algorithm79. The converted colour space reads for each time point were then mapped against the Trinity assembly. Data handling and differential expression analysis were performed as described above. The RNA-seq reads have been submitted to NCBI under Bioproject PRJNA631777.Paired-end colour space reads of each RNA-seq library were converted to nucleotides with an in-house tool. Next, de novo assembly was performed using the Pinus EST transcripts  that achieved at least eight CPM mapped counts were clustered using SplineCluster, a Bayesian model-based hierarchical clustering algorithm for time series data40. The average CPM counts of the four libraries at each time point for each transcript were first calculated. These mean CPM counts were divided by the average CPM counts across all time points. With this normalisation, the average expression level of each gene is set to 1 and the expression level can vary between 0 and 4, however the fold change between time points remains unchanged. Next, Bayesian hierarchical clustering analysis was carried out with default parameters except priorprecision and normalisetargets was set to 1e-30 and 0, respectively. Visualisation of the clusters was done using an in-house tool that used the SplineCluster data as input.The 17 584 Pinus EST collection was obtained and complexity of obtained GO terms was reduced using Blast2GO80. GO enrichment analysis for differentially expressed transcripts in each cluster was performed as before25. Enriched GO lists were summarised and visualised in semantic similarity-based scatterplots using REVIGO41 with the allowed similarity parameter set to 0.5 and other parameters as default.Gene Ontology (GO) terms of PIANO R package27. The logarithm of fold change value, the p-value and the GO terms for 4595 differentially expressed transcripts across all time points were used as input in a PIANO R session. The gene set analysis was carried out with default parameters. The GSA results were visualised in a gene set network plot in distinct-directional class with significance level at p < 0.005.Gene set analysis (GSA) was performed with the Supplementary Figure.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.Supplementary Table 7.Supplementary Table 8.


We also observed association of plant oil consumption with increased triiodothyronine levels in HT patients , and, association of olive oil consumption with decreased systolic blood pressure  in HT patients on levothyroxine (LT4) therapy. Analysis of food consumption between HT patients with and without LT4 therapy suggest that patients do not tend to modify their diet upon HT diagnosis in our population. Our study may be of relevance to nutritionists, nutritional therapists and clinicians involved in developing dietary recommendations for HT patients.Food is considered as important environmental factor that plays a role in development of Hashimoto's thyroiditis (HT). The goal of our study was to identify food groups, assessed by food frequency questionnaire, that differ in consumption frequency between 491 patients with HT and 433 controls. We also analysed association of food groups with the wealth of HT-related clinical traits and symptoms. We found significantly increased consumption of animal fat (OR 1.55,  HT is a chronic autoimmune thyroid disorder (AITD) characterized by the production of thyroid autoantibodies against thyroid peroxidase (TPOAb) and thyroglobulin (TgAb)2. Other main characteristics of HT include infiltration of lymphocytes and ruination of thyroid tissue, which usually leads to hypothyroidism3. This disease predominantly affects female population and is considered as one of the most frequent endocrine disorders4. The estimated incidence of HT is 350/100,000 for females and 80/100,000 per year for males5. It is most often diagnosed in women between the age of 30 and 606.Environmental factors, in addition to genetic factors, play important role in Hashimoto’s thyroiditis (HT) development7, especially in areas with sufficient iodine supply, is one of the most known factors that increase the incidence of HT8. Also, low vitamin D levels (below 30 ng/mL)9 and low selenium intake (bellow 40 µg/day)10 are proposed to be risk factors for HT development, albeit a role of these factors in HT still needs to be confirmed. Conversely, alcohol intake was proposed to decrease the risk for HT11. Other dietary factors are also considered as potential environmental modifiers of the clinical appearance of HT15. A diet low in gluten has been recently proposed to have beneficial effects on the disease course in a study of 34 women with HT13. Another study of 83 patients with HT indicated that restriction of lactose intake in lactose-intolerant HT patients resulted with decreased thyroid-stimulating hormone (TSH) levels14. However, the current knowledge of this topic is scarce and only the following two studies analyzed the association between thyroid function and dietary factors using food frequency questionnaire (FFQ) as an assessment tool to date. A large population-based study of 65,981 participants found an association of the vegan diet with a lower risk of self-reported hypothyroidism16. Another recent study showed that higher animal fat and butter intake are associated with the risk of positivity to TPOAb and/or TgAb antibodies using 1,887 participants, whereas diet rich in vegetables, dried fruit, nuts and muesli consumption decreased that risk17.High iodine intake (median urinary iodine concentration ≥ 300 μg/L)The first goal of our study is to identify specific food groups, assessed by FFQ, that differ in consumption frequency between patients with HT and control subjects. Our second goal is to perform a comprehensive set of analyses to test associations of diet with the wealth of HT-related phenotypes and symptoms. Our third goal is to exploit if HT patients change their diet upon HT diagnosis. The overall aim of our study is to contribute to the knowledge related to diet and HT and may be of relevance to nutritionists, nutritional therapists and clinicians involved in developing dietary recommendations for HT patients.18. The study involved newly diagnosed patients along with patients on follow up. Diagnosis was made by a clinical specialist in nuclear medicine primarily based on clinical examination and thyroid ultrasound. Only individuals with echographic pattern of diffuse thyroid disease (unhomogenous thyroid tissue with diffusely reduced echo levels) were included in the study. Diagnosis of HT was further complemented by evaluation of biochemical measurements. HT was confirmed by at least one of the laboratory parameters falling out of referent ranges: increased TSH and/or decreased thyroid hormones  and/or increased TPOAb and TgAb autoantibodies. Hormones  and levels of thyroid antibodies (TgAb and TPOAb) were measured from the plasma of participants by immunoassay using the fully automated "Liaison" Biomedica Chemiluminescence Analyzer . We used the following reference values for hormones and thyroid antibodies levels that are valid for our population: TSH (0.3–3.6 mUI/L), T3 (1.3–3.6 nmol/L), T4 (57.4–161 nmol/L), fT4 (10.1–22.3 pmol/L), TPOAb (1–16 IU/mL), TgAb (5–100 IU/mL). Thyroid ultrasonography was performed using the Medison Accuvix V10  high frequency linear probe (8–12 MHz). Thyroid volume was calculated as a sum of volumes of both lobes, where the volume of each lobe was calculated as length × width × depth × 0.47919. The body surface area (BSA) in m2 was calculated as W0.425 × H0.725 × 0.007184, where W is the weight (kg) and H the height (cm).Our study investigated 924 individuals from the region of Split (Southern Croatia), including 491 HT cases and 433 control participants. All procedures have been established according to guidelines of the most relevant thyroid societies . Patients were recruited from the Outpatient clinic for thyroid disorders at the University Hospital Split in the period from 2013 to 2017 and are a part of the Croatian Biobank of patients with Hashimoto’s thyroiditis (CROHT)21.Our HT cases include 289 HT patients without LT4 therapy and 177 HT patients who are taking LT4 therapy (information on LT4 therapy was not available for 25 HT patients). HT patients without LT4 therapy had median TSH levels just above the upper referent values and T3 and T4 levels in normal ranges, indicating that these patients were newly diagnosed or in follow-up care with preserved thyroid function. HT patients with LT4 therapy had median TSH, T3 and T4 levels within the reference ranges, suggesting that their LT4 supplementation was appropriate. Median TgAb/TPOAb levels were increased  in both sub-groups of patients. Clinical and sociodemographic characteristics of HT cases are shown in Table 22. We selected individuals from the region of Split to match the origin of HT cases. We used all available self-reported phenotype information to exclude individuals from controls that have HT or any other type of thyroid disorder, such as Graves’ disease, thyroid cancer, non-autoimmune hypothyroidism or individuals that used drugs for any type of thyroid disorder. We also used available laboratory measures on thyroid hormones levels  to keep only individuals whose hormone values fall within the reference range for our population. Finally, we excluded from controls all individuals with positivity to TPOAb (TPOAb level > 16 IU/mL) or TgAb (TgAb level > 100 IU/mL). In this way, we strengthened our control group by minimizing the possibility of having individuals with undiagnosed AITD or any other type of thyroid disorder. Clinical and sociodemographic characteristics of controls are shown in Table Control participants were derived from the population-based “10,001 Dalmatians project” cohort that is a part of the Croatian Biobank program with a large phenotype information on study participantsWritten informed consent was obtained from all study participants. The study was approved by Ethics Committees from the University of Split, School of Medicine  and University Hospital Split . Both Ethics Committees declared that the study is in accordance with the provisions of the Code of Ethics and the Helsinki Declaration.23. Dietary intake of HT patients was assessed using the FFQ that consisted of 51 items concerning foods and beverages. The frequency of intake of each food item was measured using six categories: every day, 2–3 times a week, once a month, once a week, rarely, and never. Participants reported the frequency of consummation of specific food but did not quantify the amount of consumed food (servings). Dietary intake in control participants was assessed using the FFQ that consisted of 54 items regarding foods and beverages. The frequency of food intake was measured using five categories: every day, 2–3 times a week, once a week, rarely, and never. Additionally, there was a question in both FFQs regarding fat consumption with three choices  and three frequency categories . Prior analysis, we grouped 48 food items that were common in both questionnaires into 22 food groups  differ in dietary habits, as this analysis is important for data interpretation. The rationale behind this analysis was to take into consideration possible changes in diet between these two sub-groups  which could introduce spurious associations for our main case–control analysis. We used a logistic regression model, where therapy status (with/without LT4 therapy) was used as the dependent variable and 22 food groups as independent variables, along with age and gender.p values corrected for 9 tests (p < 0.0056) as statistically significant.We also tested the association between weekly intake of 22 food groups and HT-related quantitative traits  using a linear regression model, where a selected quantitative trait was the dependent variable and 22 food groups were independent variables. Normality of distribution of residuals was tested using Kolmogorov–Smirnov test while homogeneity of variance was tested using Levene's test. In each model, we included age, gender, and physical activity  was considered statistically significant. All statistical analyses were performed using the Statistical Package Software for Social Science, version 16 .Finally, we assessed the association between 22 food groups and 16 symptoms of hypothyroidism in the sub-group of HT patients without LT4 therapy. For each symptom, we used a logistic regression model where symptom status (with/without symptom) was used as the dependent variable and food groups as independent variables. Age, gender, physical activity, body mass index (BMI) and TSH levels were also included as covariates. The p < 0.0001], processed meat  and nuts  were significantly more frequently consumed in HT patients than in controls, whereas red meat , non-alcoholic beverages , whole grains , plant oil , olive oil , liquor , oily fish  and fruits  were significantly less frequently consumed in HT patients than in control participants , nts Fig. . Distribp = 0.003] , 03] Fig. .Figure 2p < 0.0001) and in the sub-group of HT patients that were on LT4 therapy . We also found a negative association between the consumption of olive oil and systolic blood pressure in the sub-group of HT patients that were taking LT4 therapy . All other close-to-significant observations are reported in SI Table Linear regression analysis between weekly intake of 22 food groups and HT-related phenotypes detected a significant positive association between plant oil consumption and T3 levels in the group including all HT patients , 15, we performed a set of analyses to detect food groups that: (a) differ between HT patients and controls and (b) may be associated with clinical aspects of HT. We found animal fat, processed meat and nuts, as more frequently consumed food groups in HT patients than in controls. Conversely, red meat, non-alcoholic beverages, whole grains, plant oil, olive oil, liquor, oily fish, and fruits were less frequently consumed in HT patients than in controls. Furthermore, this study detected a positive association between plant oil consumption and T3 levels in HT patients and a negative association between olive oil consumption and systolic blood pressure in the sub-group of HT patients that were on LT4 therapy. Another interesting observation of this study is the lack of association between the 16 symptoms of hypothyroidism with food groups, with the exception of the positive association between fruit consumption and constipation in the group of HT patients without LT4 therapy. Finally, we do not observe that patients with LT4 therapy are prone to change their diet due to disease presence. In the text below, we will discuss observed significant associations according to food group.As dietary habits may play a role in HT disease development17, key characteristics of HT2. Triglycerides in animal fats, mainly contain saturated (SFA) and monounsaturated fat acids (MUFA)26. It is known that SFAs may induce development and affect progression of many chronic diseases through inflammatory response27. Two recent studies showed that high-fat diet causes thyroid dysfunction in rats and also induces hypothyroidism by decreasing total T4 and fT4 with increasing TSH levels29. These studies suggested that excess intake of animal fat contributes to pathogenesis of hypothyroidism. Our results are in line with these studies, although this result should not be taken as a suggestion to restrict animal fat consumption until further interventional studies are performed.The most significant result of our study was observation of significantly increased consumption of animal fat in HT patients in comparison to controls Fig. . It is i26. Other ingredients of processed meat include nitrates and nitrites, which are used as additives for curing meat products, together with sodium30. A recent systematic review on animal studies showed that high exposure to dietary nitrate or nitrite induces histomorphological changes in thyroid tissue and a decrease in serum levels of thyroid hormones31. It is suggested that high exposure to nitrate/nitrite could inhibit iodine uptake by binding to the sodium-iodide symporter on the surface of thyroid follicles33.Processed meat in our study  consists mostly of fat, then proteins and in very small amount carbohydrates35, from which both of our cohorts (HT patients and controls) were derived. More specifically, a comparison of daily nuts consumption between two population-based cohorts from Split, one recruited in 2008–2009 and the other in 2012–2013, showed an increase in nuts consumption from 7 to 11%, respectively35. Our control group was formed from the former population-based study whereas our HT case cohort was formed during 2015–2016. Therefore, an observed increase of 2.3% in nuts consumption between HT cases and controls in our study may purely reflect the generally established trend of increase in nut consumption in our population. It is important to state that our FFQ did not distinguish between different types of nuts . Due to all abovementioned reasons, the interpretation of results regarding nuts is difficult and should be taken with caution.We believe that increased consummation of nuts in HT cases may be false positive finding. We base this statement on the recently observed trend of increased nut consumption in the city of Split36, iron, and zinc37, which are essential for the normal function of thyroid hormone synthesis38. Additionally, red meat is rich in vitamin B1237 whose deficiency is associated with AITD39. Unlike processed meat, fresh red meat contains less fat26 and does not have additives . However, for the purposes of our analyses, red meat included several types of meat  that may contain various amounts of animal fat, which is a limitation factor for interpretation of results. An important remark, we have compared intake of food groups between two sub-groups of HT patients (those with and without LT4 therapy) and found unequal red meat consumption  in 50% of recommended daily allowances (RDA). There is literature record of vitamin deficiencies  in patients with thyroid diseases. An anti-inflammatory diet, rich in vitamins and minerals, is recommended as a diet therapy for HT43. Consumption of whole grains, and gluten-rich refined grains, was significantly higher in controls than in HT cases. This is an important observation because it does not support the proposition that a gluten free diet is beneficial to HT patients, as is usually recommended45. In line with these results, a recent study showed that muesli consumption, which consists mainly of whole grains46 was associated with a decreased risk of positivity to TPOAb or TgAb antibodies17. Further large randomized studies are needed to give a conclusive answer about effects of gluten reduction in HT patients.Whole grains are rich in dietary fibers, source of energy for cecum and colon-residing microbiota that metabolize complex carbohydrates and also produce short-chain fatty acids (SCFAs), which are all important for metabolism, cell proliferation and immune system47. Sunflower oil, the most frequently used type of refined plant oil in Croatia, is a natural source of powerful antioxidant vitamin E48.Plant oil, especially cold pressed oil, such as sunflower oil, pumpkin seed oil, and olive oil are rich in bioactive components (polyunsaturated fatty acids (PUFA), tocopherols and various phenols) that are associated with anti-inflammatory properties and reduction of oxidative stress50. Oleocanthal is a natural anti-inflammatory compound from extra virgin cold pressed olive oil that has ibuprofen-like activity51. In fact, many studies have reported anti-inflammatory and immunomodulatory effects from the regular consumption of olive oil54.Olive oil, rich in phenols and oleic acid, was suggested as a dietary factor that may induce clinical benefits in some autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis55.Moderate alcohol consumption was already proposed to decrease the risk for HT, which is also in line with our results59. Importantly, a recent study reported an association between omega-3-rich oily fish intake and decreased serum thyroid autoantibodies levels during pregnancy and suggested dietary prophylaxis against thyroid-related postpartum problems60. Oily fish and other seafood are also good source of selenium, iodine, iron and zinc61 which are known that could play a beneficial role on a AITD63.Oily fish consumption or supplementation with omega-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may reduce inflammation and are of clinical relevance for various autoimmune disorders64.Finally, beside already mentioned beneficial roles of vitamins, minerals and dietary fibers, fruits additionally contain phytochemicals such as polyphenolic compounds that are known for their anti-inflammatory and antioxidant effects on human health65, as our main resource, and United States66 (Mediterranean-Style Eating Pattern), a lower intake of animal fat and processed meat is identified as healthy eating patterns. Although animal fat is rarely consumed in HT patients  in controls. Our study also showed that HT patients are not prone to change their diet upon disease diagnosis, as we have found that two sub-groups of HT patients (depending on LT4 therapy) have no main differences in dietary habits, except for red meat consumption. None of the scientific societies have made dietary recommendations for patients with HT, yet these are highly awaited and still need to be developed. Our study contributes to this aim and increases the, currently limited, knowledge related to diet and HT. Our findings may be of particular relevance to nutritional therapists and clinicians involved in treatment of patients with HT.Supplementary information


FROH), homozygosity (FHOM), genomic relationship matrix (FGRM) and pedigree (FPED). Furthermore, we identified genomic regions with high ROH frequencies and annotated their candidate genes.Runs of homozygosity (ROHs) are homozygous segments of the genome where the two haplotypes inherited from the parents are identical. The current availability of genotypes for a very large number of single nucleotide polymorphisms (SNPs) is leading to more accurate characterization of ROHs in the whole genome. Here, we investigated the occurrence and distribution of ROHs in 3,692 Large White pigs and compared estimates of inbreeding coefficients calculated based on ROHs (Sus scrofa chromosomes (SSC) by ROH were on SSC14 and SSC13, respectively. The highest pairwise correlation among the different inbreeding coefficient estimates was 0.95 between FROH_total and FHOM, while the lowest was − 0.083 between FGRM and FPED. The correlations between FPED and FROH using four classes of ROH lengths ranged from 0.18 to 0.37 and increased with increasing ROH length, except for ROH > 10 Mb. Twelve ROH islands were located on four chromosomes . These ROH islands harboured genes associated with reproduction, muscular development, fat deposition and adaptation, such as SIRT1, MYPN, SETDB1 and PSMD4.In total, 176,182 ROHs were identified from 3,569 animals, and all individuals displayed at least one ROH longer than 1 Mb. The ROHs identified were unevenly distributed on the autosomes. The highest and lowest coverages of FROH can be used to accurately assess individual inbreeding levels compared to other inbreeding coefficient estimators. In the absence of pedigree records, FROH can provide an alternative to inbreeding estimates. Our findings can be used not only to effectively increase the response to selection by appropriately managing the rate of inbreeding and minimizing the negative effects of inbreeding depression but also to help detect genomic regions with an effect on traits under selection. The inheritance of identical haplotypes from a common ancestor creates long tracts of homozygous genotypes known as runs of homozygosity (ROHs) , 2. LongF), which is defined as the probability that both alleles at any locus within an individual are IBD [F. Traditionally, F has been estimated based on pedigree information (FPED). Hence, the estimation of FPED relied strongly on the accuracy and amount of pedigree data available [FGRM) using genome-wide single nucleotide polymorphism (SNP) data [F can also measure homozygosity (FHOM) directly and, thus, can accurately reflect the actual percentage of the genome that is homozygous. Furthermore, genomic F allows us to estimate inbreeding and inbreeding depression for specific genomic regions, which is not possible with FPED. In addition, genomic F can be estimated in populations without pedigree information [On the basis of levels of autozygosity, ROHs can be used to estimate the inbreeding coefficient of an individual ( are IBD . Inbreed are IBD . Since ivailable . Currentvailable . The inbNP) data . Genomicormation .F using genotypic data, there is a need to distinguish between alleles that are IBD or identical by state (IBS). This distinction is not easy to make when single markers are analysed, but the use of ROH allows it. Hence, FGRM and FHOM always overestimate inbreeding levels compared to FPED [When computing the more accurate genomic  to FPED , and an FROH) and estimated their correlations with those from other methods, including genomic relationship matrix (FGRM), homozygous coefficients (FHOM) and pedigree-based coefficients (FPED). Moreover, ROH islands may have occurred due to selection for functionally important traits.The objective of this study was to identify and characterize ROH patterns in a Large White pig population. We further computed the inbreeding coefficients based on the ROHs identified  .Sus scrofa 10.2 genome version. To update the SNP positions, we reordered the SNPs according to the newest version of the pig genome, Sus scrofa 11.1. Following this, 34,689 autosomal SNPs remained, with a mean distance of 67.052 kb between adjacent SNPs.In this study, ear tissue samples were collected from 3,692 Large White pigs from two commercial companies. In these two companies, there were 1,466 and 2,226 pigs, respectively. All samples were genotyped with the GeneSeek GGP Porcine HD array. The SNP chip comprises 50,915 probes that are distributed across 18 autosomes and two sex chromosomes according to the Quality control was performed using PLINK v1.90 software  accordinNe) was calculated from linkage disequilibrium (LD) [The effective population size (ium (LD)  using thNT(t) is the effective population size estimated t generations in the past, ct is the recombination rate defined for a specific physical distance between SNP markers t generations in the past, f(ct) is the Haldane mapping function of the genetic distance in Morgans between SNPs, where mentclasspt{minimaNe was computed by implementing SNeP software [Ne estimation were 0.05 and 5 Mb, respectively. The other parameters were set by default [In this study, software . Considesoftware , we usedsoftware . The min default .ROHs were identified for each individual using PLINK v1.90 software , which uns is the number of SNPs per individual, ni is the number of individuals, and where α is the percentage of false positive ROHs (set to 0.05 in the present study), In this study, the ROHs identified were further divided into three classes: 1~5, 5~10 and > 10 Mb.F) were used for all animals:FPED) were computed for 3,569 pigs using pedigree information recorded between 2011 and 2019. For all individuals who passed quality control, complete pedigree records were available for 3 to 10 generations with an average depth of 6.86. Pedigree information on a total of 7,572 animals between 2011 and 2019 was available. The FPED was estimated for datasets  according to Wright’s coefficient [pedigreemm [The genealogical inbreeding coefficients (fficient  with thedigreemm .FHOM) was computed as FHOM = (O − E)/(L − E), where O is the number of observed homozygotes, E is the number expected by chance, and L is the number of genotyped autosomal SNPs.Genomic inbreeding based on homozygous SNPs was determined using PLINK v1.90 software . The inbFGRM) estimates were calculated by GCTA software [FGRM was calculated as xi is the number of copies of the reference allele for the ith SNP, m is the number of SNPs, and pi is the frequency of the reference allele.Genomic SNP-by-SNP inbreeding coefficient (software . The FGRFROH). The FROH for each animal was calculated as i of individual i, and Lauto is the autosomal genome length covered by the SNPs included in the chip.Genomic inbreeding coefficients were also estimated based on ROHs . The biological function of each annotated gene within the ROH islands was determined through an extensive accurate literature search.The gene content of the ROH islands was annotated using the annotation database provided by NCBI (Ne) estimated based on linkage disequilibrium (LD) is illustrated in Fig. Ne from 197 generations to 10 generations ago of each breed was estimated based on the LD decay. An increasing Ne as a function of the number of generations was observed, with a Ne of 99 estimated at 10 generations ago and 288 estimated at 197 generations ago.The tendency of effective population sizes  obtained using all pedigree data available ranged from 0 to 0.156 with an average of 0.011. The four kinds of FROH were calculated based on three classes of ROH and the total ROH lengths. The average genomic inbreeding based on the total observed ROHs  was 0.140 with a range from 0.008 to 0.409 in this population. The estimated FGRM inbreeding coefficients ranged from −0.168 to 1.359 (mean = 0.099), and the estimated FHOM inbreeding coefficients ranged from −0.177 to 0.431 (mean = 0.103).In the present study, seven kinds of inbreeding coefficients were estimated based on the pedigree or genomic data of all individuals. The pedigree-based inbreeding estimates .Figure SIRT1 gene. SIRT1 is related to porcine ovarian cell function, suppresses adipogenesis [MYPN (63.21% of occurrences). The MYPN gene has been documented to be associated with meat and carcass traits in Italian Large White pigs [The percentage of SNPs in ROHs was plotted against the positions of the SNPs along the chromosomes in Fig. ogenesis , 22, andogenesis . The regite pigs . Twelve FPED value is the statistical expectation of the probable IBD genomic proportion [FPED does not take into account the stochastic events of recombination during meiosis [FPED could not completely show the actual relatedness among individuals in the population. With the development of high-throughput genotyping technologies and the reduction in genotyping costs, the inbreeding coefficient can be computed based on molecular information [FHOM cannot distinguish IBD alleles from IBS alleles, it might overestimate inbreeding levels [FHOM and FGRM values can be negative for some individuals. Therefore, using the above three methods to estimate the inbreeding coefficient is not sufficiently accurate. FROH can alleviate the issues mentioned above, and thus, FROH might be a more effective and accurate alternative for quantifying animal relatedness and inbreeding levels in theory [Traditionally, the inbreeding coefficient was estimated based on pedigree data. In reality, pedigree information might be erroneous, such as having a missing parent or incorrect parent information. Furthermore, Large White pigs in the base population could be hypothesized to be unrelated. Moreover, the oportion , and FPE meiosis . Thus, Formation –29. Geneormation . Since Fg levels . In addin theory , 32.FROH in the Large White pig population. The correlation between the FPED and FROH estimates was low (0.18–0.37) (Table FROH > 10 Mb and FPED was the highest (0.37) among those between different classes of ROH and FPED in the Large White pig population, while the correlation between FPED and FROH_total was slightly lower (0.35)  Fig. . Compareg Morgans, where g is the number of generations since the common ancestor. Recombination events can interrupt long chromosome segments, so long ROHs (~ 10 Mb) arise as a result of recent inbreeding (up to five generations ago), and short ROHs (~ 1 Mb) are produced by IBD genomic regions from old ancestors [ROHs can reveal the time that inbreeding occurred given the approximate correlation between the length of a ROH and the distance to the common ancestor due to the occurrence of recombination events over time. Fisher  reportedncestors , which aROH than with GGRM by simulation study.ROHs can be used to improve the accuracies of genomic breeding values (GEBV). EBVs estimated by the traditional BLUP method are based on pedigrees, which are used to construct a numerator relationship matrix (A matrix). If the A matrix is replaced by a relationship matrix based on genotype data (G matrix), GEBV can be obtained with BLUP. Based on the genotype matrix, it is not possible to identify whether IBS marker alleles are IBD or not. ROHs can identify alleles in the same or different individual(s), which indicates IBD . Luan etSIRT1 may regulate granulosa cell apoptosis during follicular atresia in porcine ovaries [SETDB1 plays an essential role in the maintenance of gonocyte survival in pigs, and knockdown of SETDB1 can induce gonocyte apoptosis [PSMD4 effectively inhibits sperm-oocyte binding [GNRHR2 is involved in regulating reproductive behaviour in pigs [CES5A, GAL3ST1 and SPAG17 are essential for spermatogenesis and male fertility [MYPN showed an association with traits related to muscularity in Pietrain×(Landrace×Large White) and Duroc × Pietrain, particularly association with ham weight and lean content in Duroc × Pietrain [SLC12A4 is differentially expressed between the white and red skeletal muscle of Chinese Meishan pigs [SIRT1 may downregulate pig preadipocyte proliferation and differentiation [FMO5 plays a role in increasing glucose metabolism and insulin sensitivity in brown adipose tissue [SIM1 is involved in the regulation of energy homeostasis [KIF1BP and MCHR2 are involved in the regulation of food intake [HORMAD1, TBX15 and WARS2 are also associated with obesity [ADGRB3 is related to environmental information processing and environmental adaptation in domestic yak [Here, the ROH islands harboured many candidate genes controlling economically important traits of Large White pigs Table S. We iden ovaries  and can  ovaries . SETDB1 poptosis . PSMD4 e binding . GNRHR2  in pigs . CES5A, ertility –46. SomePietrain . SLC12A4han pigs . SIRT1 mntiation . FMO5 ple tissue . SIM1 iseostasis , and KIFd intake , 52, whid intake . HORMAD1 obesity , 55. In stic yak .v6.8 tool [Sus scrofa annotation file as background to identify significant (P < 0.05) GO terms and KEGG pathways. Several GO terms  were significant, and two were significant for KEGG  based on run-of-homozygosity islands.


The almond supplementation did not cause gastrointestinal disturbances. Our study suggests that acute almond supplementation 2 h before exercise can improve performance in endurance exercise in trained subjects.Several studies have investigated the effects of fat intake before exercise on subsequent substrate oxidation and exercise performance. While some studies have reported that unsaturated fatty acid supplementation slightly increases fat oxidation, the changes have not been reflected in the maximum oxygen uptake or in other performance and physiological parameters. We selected almonds as a fatty acid (FA) source for acute supplementation and investigated their effect on non-esterified fatty acid (NEFA) values and exercise performance. Five physically active male subjects  were randomly assigned to take an almond or placebo supplement 2 h before participating in two cycling resistance training sessions separated by an interval of 7–10 days. Their performance was evaluated with a maximal incremental test until exhaustion. Blood samples collected before, during, and after testing were biochemically analysed. The results indicated a NEFA value average increase of 0.09 mg·dL In endurance sports, the pattern of energy substrate use changes over time, even when the exercise intensity remains constant. The longer the time spent on exercise, the greater the energy substrate contribution of fat ,2. In enTrained individuals make greater use of FAs as an energy source and perform better in endurance exercise due to their ability to increase glycogen stores and use them sparingly in submaximal efforts ,6,7,8. FPrevious studies ,16,17,18Prunus dulcis) are highly nutritional, mainly due to their high lipid content (25–66 g per 100 g−1 (fresh weight)), which also makes them highly calorific  production due to, among other reasons, improved mitochondrial respiration chain oxidation flows; ROS are produced by an electron transfer that requires a high energy input with a very short lifetime (from milliseconds to nanoseconds) . Repeate2max) or other performance and physiological parameters [Based on the assumption that unsaturated FA-rich diets and endurance exercise both have positive (if different) effects on metabolic and cardiovascular health, and given that they both increase the oxidative capacity of fats, their combination is likely to be synergistic . While srameters ,48,49,50In our experimental study, we evaluated the effect of acute FA supplementation , containing unsaturated FA and antioxidant micronutrients, on prolonged resistance training tests in laboratory conditions.−2) who perform recreational sports training 3 to 5 days a week. The exclusion criteria were: (1) current or recent injury, (2) intake of fish oil or other FA supplements, and (3) any other condition that could prevent compliance with a maximum exercise test protocol. The study was approved by the institutional ethics committee  and was performed in accordance with the Declaration of Helsinki. Before participating, the subjects read a description of the study and its risks, and signed an informed consent.The participants volunteering in the study were 5 physically active men  after taking either an active supplement or a placebo supplement. The order of supplementation was randomised using a random number generator . The subjects performed all the tests in a similar postprandial state  and were instructed not to perform intense physical activity in the 72 h before testing.The ingredients of the active supplement were 60 g of ground almonds mixed with 60 mL of milk (to obtain a paste) and 6 g of fructose (as a mild sweetener). The placebo supplement contained the same ingredients (60 mL of milk and 6 g of fructose), but with 100 g of white bread instead of almonds . The supAs indicated in 2max to be used for submaximal testing, each participant performed a maximal incremental test until exhaustion, with workload increments of 20 W per minute. The test was performed one week before the baseline test (stage 0).To identify the individual workload that corresponded to 50% of the VOTo facilitate multiple blood extractions during the experiments, a venous catheter was inserted into a superficial vein in the forearm of each subject.Stage 0. BaselineThe experimental stages  were ideSubjects took the supplement (active or placebo) and blood was sampled .Stage 1. Pre-testingBlood was sampled at minutes 30, 60, 90, and 120  after supplement intake. The subjects remained inactive during this 2 h period.Stage 2. Submaximal testSubjects performed a 1 h submaximal test at 50% loading  and blood was sampled at minutes 15, 30, 45, and 60 (every 15 min).Stage 3. Maximal testWith no rest period between Stage 2 and Stage 3, the subjects performed a maximal incremental test involving 6 min steps with power increments of 25 W  followed by 1 min recovery periods until exhaustion. Blood was sampled 5 times in the first part, i.e., at minutes 7, 14, 21, 28, and 35.Stage 4. RecoveryAfter maximal test completion to exhaustion, blood was sampled at minutes 5, 10, and 20 during recovery.The peripheral venous blood samples were biochemically analysed to determine glucose, lactate, uric acid, urea, cholesterol, TG, NEFA, high-density lipoprotein (HDL), glutamate-oxaloacetate transaminase (GOT), and glutamate-pyruvate transaminase (GPT) concentrations.Subjects were instructed not to make changes to their diet during the study period (to avoid changes that might confound the results) and to eat the same light breakfast on each of the test days.Subjects performed the tests on pre-calibrated cycle ergometers . To evaluate differences in the total work performed between the two supplementations, we used the power of each step and the time spent in each workload, assessing the total capacity of the power developed by each subject.2peak (L·min−1), relative VO2peak (mL·kg−1·min−1), minute ventilation , tidal volume , and the respiratory exchange ratio (RER) were measured breath-by-breath using a two-way mask  and an automatic gas analysis system  equipped with a pneumotachometer. The gas and volume calibrations were performed before each test according to the manufacturer’s guidelines.Absolute VODuring the tests, a 12-lead electrocardiogram was performed and the heart rates (HR) of the subjects were continuously monitored .All tests were conducted during the morning at a room temperature of 22 °C to 24 °C and a relative humidity of between 55% and 65%.® Plus system . Blood lactate was measured using a photocolorimetry system . NEFAs were analysed using a gas chromatograph  equipped with a flame ionization detector; peaks were identified based on the retention time in relation to FAME standards , and peak areas were automatically computed.Glucose, uric acid, urea, cholesterol, TG, HDL, GOT, and GPT concentrations were determined using dry chemistry analysis, performed with the Reflotront-test for paired samples was used to explore differences between the means for the variables measured on test days 1 and 2. Statistical significance was set to p < 0.05. Analyses were performed using SPSS 19.0 .Descriptive statistics were calculated for all the variables. To test the normality of the variables, the Shapiro–Wilk test was used. An analysis of variance (ANOVA) for repeated measures was performed to evaluate differences between two different supplements in the blood samples. Student’s The loadings for the submaximal test are shown in 2, RER, and HR values increased significantly (p < 0.05) compared to the placebo, but not VO2 values.The metabolic results for the submaximal test at 50% loading are summarised in t = 2.91, p = 0.043, statistical power = 0.596), reflecting a generalised improvement of 20.6%.The performance results for the maximal test (maximum loads) are summarised in Cardiorespiratory and metabolic data from the maximal tests are summarised in The biochemical data recorded at the end of the submaximal test are summarised in −1 . After active supplementation, the NEFA values remained higher compared to the placebo, with an average increase of 0.09 mg dL−1 (>30% vs. the placebo).We evaluated the impact of acute FA supplementation in the form of almonds on plasma NEFA values and endurance performance, comparing it to a placebo. Our results indicate that acute almond supplementation modified the energy substrate availability pattern in plasma. In contrast to the little increase produced by the placebo supplementation, almond supplementation before exercise significantly increased plasma NEFA concentrations during the physical test by a mean value of 0.09 mg dLNEFAs, as an oxidisable fuel for physical exercise, can improve performance. According to previous studies ,51,52,53−1 (fresh weight)), and richness in unsaturated FAs, especially oleic acid (monounsaturated) and linoleic acid (polyunsaturated). In our data, triglycerides increased 14.7% with almond supplementation with respect to placebo after submaximal exercise, with similar dietetic and exercise conditions.An important factor in fat supplementation during exercise is the type of FAs administered. It is important to bear in mind that fat digestion and absorption is a lengthy process, depending on the length of the FA chain, with long-chain TGs, for instance, being absorbed more slowly than short- or medium-chain TGs . Diet alSeveral studies have evaluated the effects of supplements made with healthy foods rich in monounsaturated FAs, such as almonds, pistachios, and extra-virgin olive oil. In a study by Nieman et al. , performThe supplements used in our study were well received by the subjects and did not cause any gastrointestinal problems. The high fat content of the supplements had no negative influence on exercise performance in our study, unlike in other studies reporting impaired physical performance due to fat intake . DigestiThe calorie difference between the active supplement  and the placebo  was 90 kcal, while the carbohydrate content was lower. The main difference was in the amount of fat. Further research is warranted to explore differences between different FAs used for acute supplementation in endurance exercise.Studies in humans have shown that the consumption of almonds increases circulating levels of α-tocopherol in a dose-dependent manner ,63 and r2 production during the submaximal effort, which could have been because there were no differences in the energy substrate used. During the maximal effort, there were also no differences in the maximum oxygen consumption reached, with more CO2 being produced. This could indicate that the positive effect on performance was due to the peripheral effect of the polyphenols rather than the energy savings from the extra lipid supplement, resulting in a reduced perception of fatigue that enabled the physical effort to last longer.No differences were observed for oxygen consumption or COThe main limitation of this study was the small number of participants and the fact that our sample was composed only of men. The complexity of the protocol, however, needed a homogeneous and trained sample to rule out any training effect. Studies with larger samples would be necessary to confirm our findings, explore individual differences in responses, and test differences arising from other factors such as gender. Since oxidative stress and inflammatory biomarkers could not be assessed in this research, we cannot evaluate the role of phenolic and polyphenolic compounds in almonds in improving the antioxidant capacity of the athletes. Therefore, further research is warranted analysing inflammatory and oxidation markers to confirm this hypothesis.Our results, in summary, suggest that almonds can be included in pre-training or pre-competition supplements for endurance athletes.The acute supplementation with almonds (60 g), administered 2 h before exercise, increases mean plasma NEFA values by 30% and improves exercise performance by 20.6%. Our study suggests that almond supplements could have a positive effect on performance in endurance exercise. Further studies are required to confirm the effects on long-term exercises, using it directly in field tests with larger samples and other population groups .