Sustainable food security is a major challenge in today’s world, particularly in developing countries. Among many factors, environmental stressors, i.e., drought, salinity and heavy metals are major impediments in achieving sustainable food security. This calls for finding environment-friendly and cheap solutions to address these stressors. Plant growth-promoting rhizobacteria (PGPR) have long been established as an environment-friendly means to enhance agricultural productivity in normal and stressed soils and are being applied at field scale. Similarly, pyrolyzing agro-wastes into biochar with the aim to amend soils is being proposed as a cheap additive for enhancement of soil quality and crop productivity. Many pot and some field-scale experiments have confirmed the potential of biochar for sustainable increase in agricultural productivity. Recently, many studies have combined the PGPR and biochar for improving soil quality and agricultural productivity, under normal and stressed conditions, with the assumption that both of these additives complement each other. Most of these studies have reported a significant increase in agricultural productivity in co-applied treatments than sole application of PGPR or biochar. This review presents synthesis of these studies in addition to providing insights into the mechanistic basis of the interaction of the PGPR and biochar. Moreover, this review highlights the future perspectives of the research in order to realize the potential of co-application of the PGPR and biochar at field scale. Recently in agro-ecosystems, soil amendments are used to support plant growth and development, especially by adding organic and inorganic nutrients to the soil. Soil amendments are elements that are added to the soil to improve its ability to support plant life . Soil amHowever, the application of organic soil amendments to agricultural soils poses a number of threats to the agro-ecosystem and human health. Organic soil amendments often include a range of pollutants, including heavy metals, potential human pathogens, persistent organic pollutants, and emerging pollutants. From the emerging pollutants the presence of antibiotic-resistant bacteria, antibiotic residues, and antibiotic-resistant genes in agricultural organic amendments is of great concern at the moment, due to the harmonious risks to human health . Soil amVarious PGPR have been isolated and proven to alleviate various environmental stresses in plants and boost productivity. They may improve soil quality and boost plant productivity by direct and indirect mechanisms. Nitrogen fixation, phosphate and potassium solubilization, and production of growth-promoting phytohormones like indole acetic acid and siderophores are direct mechanisms through which PGPR perform these aforesaid functions; whereas, the indirect mechanisms involve production of lytic enzymes and antibiotics, lowering the soil pH, production of exopolysaccharides, etc. Fig. . The effBiochar, a char produced by pyrolyzing organic materials particularly wastes under limited oxygen supply, has gained immense popularity for its vast range of uses like enhancing soil quality, soil carbon sequestration, adsorption and mitigation of organic and inorganic pollutants from aqueous and soil media, animal feedstock, etc. Multiple review articles and meta-analyses have summarized the positive effects of biochar on soil quality and agronomic productivity as well as the factors that contribute to the ameliorative role of biochar . The bioSustainable food security is a major challenge in today’s world, more so in developing countries. The teeming millions in developing world, e.g., South Asia, South East Asia, and Africa, coupled with all around climate changes affecting agricultural operations and productivity are a major risk to sustainable food security . AccordiDifferent strategies are used to improve soil quality and increase the crop yields including land reforms, better water management, stress-tolerant varieties, increasing use of fertilizers, improved seeds, use of pesticides, genetically modified crops, plant growth regulators, and soil amendments; PGPR, biochar , 23–25. The agricultural productivity is reduced by different abiotic stresses such as salinity, drought, and heavy metal contaminants in soils among others . The worCrop and livestock production are water-intensive enterprises because agriculture is the largest consumer of water globally, accounting for 70% of global water returns . AgriculAnother important abiotic stress is heavy metals in soils resulting in losses of agricultural productivity. Due to various natural and human activities, significant amounts of heavy metals are regularly added to the soil worldwide . More thRecently, PGPR and biochar have been co-applied in various studies in order to improve soil quality and agronomic productivity under normal and stressed conditions. The explicit or underlying assumption in these studies has been that the biochar would increase nutrient availability and provide conducive habitat for the PGPR to flourish and in response the latter would perform their designated functions  at higher rates. These studies have been performed in stress-free as well as stressed soils. However, these studies have not been comprehensively synthesized and critically reviewed. This review paper aims to fill this gap. Moreover, we also present the future directions of research in order to optimally exploit the combined potential of PGPR and biochar for sustainable agro-ecosystem.Soil quality is a complex concept. The soils perform a variety of ecosystem services, which lead them to be defined from the point of view of those services . From coVarious PGPRs co-applied with biochar are proposed as a good strategy to improve soil quality –41. The Paenibacillus polymyxa and Bacillus amyloliquefaciens) showed 87% higher soil nitrate content than nitrogen only treatment [4+-N contents in PGPR + biochar and biochar + nitrogen treatments was 136.83% and 82.07% higher than nitrogen only treatment. Jabborova et al. [Bradyrhizobium japonicum and Pseudomonas putida) and different levels of maize biochar (1% and 3%) on soil nutrients. They found that co-application of the PGPR with 3% maize biochar increased available nitrogen, phosphorus, and potassium by 73%, 173%, and 17%, respectively, when compared to the 3% maize biochar only treatment. Ren et al. [Bacillus megaterium (a nitrogen-fixing bacteria) with wheat-derived biochar increased nitrate, inorganic nitrogen, and total potassium in PGPR + biochar treatment by 68%, 45%, and 21%, respectively, than PGPR only and by 22%, 16%, and 30%, respectively, than biochar only treatment. Similarly, a PGPR Bacillus megaterium, when co-applied with biochar derived from agricultural waste, was found to increase organic carbon, available phosphorus, and available nitrogen by 16%, 79%, and 15%, respectively, in comparison to the control (no PGPR and no biochar) treatment. Saxena et al. [−1 shoot in soil treated with PGPR (Bacillus sp.) co-inoculated with biochar, which was significantly higher than that in sole applications of Bacillus sp. (1.24 1.64 mg N g−1 shoot) or biochar (1.31 mg N g−1 shoot). Overall these studies indicate that co-application of biochar and PGPR works in synergy to raise the nutrient level higher than the individual application of any of these. Biochars are rich in macro- as well as micro-nutrients. When applied to soils, they contribute nutrients to soils as a result of dissolution and decomposition under the influence of soil conditions and microbial activity [A number of studies have assessed the effect of co-application of PGPR and biochar on soil quality defining physicochemical and biological properties of soils Table . Co-applreatment . Moreovea et al.  evaluaten et al.  found tha et al.  found thactivity . The PGPactivity . Consequactivity .Table 1EBacillus megaterium, along with wheat-derived biochar increased soil WHC by 24% and 18% than PGPR only and biochar only treatments, respectively [The biochar has potential to improve water holding capacity of soils, particularly for coarse-textured ones, thanks to its large surface area-to-volume ratio. A number of reviews have compiled studies on this question , 58. Somectively . Althougectively . Howeverectively , 60. It Pseudomonas sp. increased root colonization by arbuscular mycorrhizal fungi when compared to sole addition of Pseudomonas sp. and/or AMF. The phosphate-solubilizing Pseudomonas sp. enhanced available phosphorus in the soil presumably by solubilizing it from the biochar thereby leading to enhanced root colonization and overall growth of the plant [Alcaligenes sp. strain in interaction with a maize-stalk-derived biochar increased the population of soil bacteria by 30% when compared to sole application of Alcaligenes sp. and by 15% when compared to biochar only treatment. Similarly, inoculation by Bacillus megaterium of a eucalyptus plantation along with addition of wheat-derived biochar significantly improved the microbial community in the soil, thereby leading to improved nutrient availability. The authors attributed this increase in beneficial microbes to the enhanced soil organic matter content and its decomposition due to interactive effect of biochar and the inoculant [Many physicochemical properties of soil are improved by biochar, which ultimately facilitate the working of indigenous soil microbial communities. For instance, biochar may improve water holding capacity, pH (liming effect), and substrate and nutrient availability, which may lead to increase in microbial biomass, abundance and diversity , 82. Howhe plant . The comnoculant .Bacillus deuterium increased soil sucrose activity to 4.8 mg.g−1 and 3.31 mg.g−1 from 2.48 mg.g−1 in PGPR only treatment [Paenibacillus polymyxa and Bacillus amyloliquefaciens with biochar (2% w/w) treatment than that in the sole PGPR and biochar treatments, respectively [Bradyrhizobium japonicum and Pseudomonas putida) with 3% maize biochar than PGPRs only and PGPRs with 1% maize biochar. Similarly, synergistic use of Bacillus subtilis with cotton-derived biochar was found to significantly enhance the invertase and catalase activities in soil than the biochar only treatment [B. japonicum and P. putida with the biochar (10 t ha−1) has been reported to increase the activity of different enzymes like FDA activity, alkaline phosphomonesterases and proteases in the soil than biochar only and the uninoculated control  [The potential beneficial effects of combined application of biochar and PGPR have also been assessed and reported by studying various intra- and extracellular enzymes. Combined application of a biochar with a nitrogen-fixing reatment . Soil urectively . Jabboroectively  found threatment . Co-applbiochar) . Overallbiochar) , 50. It biochar) . Given tMicrococcus yunnanensis, when co-applied with 2% biochar, increased the yield to 42.1 g pot−1 from 38.9 g pot−1 when applied alone or from 36.3 g pot−1 when biochar was applied alone [Micrococcus yunnanensis only and 8% increase in phosphorus uptake than the 2% biochar alone treatments. Yuan et al. [Paenibacillus polymyxa and Bacillus amyloliquefaciens) strains with 2% biochar derived from millet straw and nitrogen fertilizer. They recorded 32.45%, 10.44% and 45.69% higher yield in PGPR + biochar + nitrogen than nitrogen only, biochar + nitrogen and PGPR + nitrogen treatments, respectively. Jabborova et al. [Bradyrhizobium japonicum and Pseudomonas putida with 3% maize biochar than in 3% biochar only treatment. Similarly, combined application of Alcaligenes sp. with 0.5 t ha−1 maize biochar enhanced the shoot fresh biomass, shoot dry biomass, plant height, grain yield, and 1000-grain weight by 9, 12, 6, 14 and 5%, respectively, than PGPR alone [Bacillus thuringiensis, Pseudomonas putida and Klebsiella variicola PGPR strains) in combination with biochar than the biofertilizer only [Enterobacter, Pseudomonas, Azospirillum, Agrobacterium and biochar raised the wheat yield to 5.04 t ha−1 than 2.56 t ha−1 PGPR only and 3.16 t ha−1 in biochar only treatments [Bacillus sp with biochar in French beans increased shoot biomass from 2.34 g pot−1 to 3.22 g pot−1, root length from 13.33 cm to 14.88 cm, and root biomass from 1.31 g pot−1 to 1.85 g pot−1, respectively [2 fixation leads to higher plant growth and yield. Indirectly, the presence of biochar may facilitate the survival of the PGPR in higher numbers in addition to providing them nutrient rich substrate thereby leading enhanced performance by the PGPR ultimately resulting in higher plant production [Sustainable agriculture requires that crops grow with a low rate of agrochemical application possessing better nutritional values and disease resistance. Widespread use of expensive agrochemicals in agriculture has led to the use of more sustainable alternatives, such as PGPR and biochar in recent decades , 90. Boted alone . Co-appln et al.  reportedPR alone . A 3% inzer only . Combinaeatments . Similarectively . Overalloduction .The PGPR are known since long to help alleviate multitude of environmental stressors that hamper plant growth and development. They have been proven very effective against drought, salinity, heavy metal contamination Fig. .Fig. 2EfFor instance, the potential of PGPR to secrete exopolysaccharides under dry conditions help induce drought tolerance in plants . Under s+ uptake due to Na+’s transient binding on sorption sites on biochar alleviate soil salinity stress for plants in biochar-amended soils [Biochar has also been shown to enhance salinity tolerance, alleviate drought stress, and mitigate the toxicity induced to plants by inorganic and organic soil pollutants. Drought stress alleviation in biochar-amended soils occurs through enhanced water holding capacity thanks to large surface area-to-volume ratio of biochar . Similared soils . Sorptioed soils , 99, 100Serratia odorifera to maize, when moisture content was 50% of the field capacity, significantly improved pH by 7 and 5%, EC by 34 and 13%, nitrate by 57 and 34%, phosphorus by 54 and 49%, extractable K by 30 and 15%, and organic matter by 69 and 21% in comparison to biochar alone and PGPR alone treatments, respectively [Cellulomonas pakistanensis or Sphingobacterium pakistanensis with biochar to Vicia faba growing on induced drought stress. They found that the combined application increased the water-use efficiency by 43.62%. In another study, soil moisture content was significantly higher in combined application of Pseudomonas sp. and biochar derived from poplar saw dust than sole application of PGPR or biochar [The combined use of PGPR and biochar perform multiple functions in alleviation of drought stress thereby leading to improved soil quality Table . Both seectively . Similarectively  co-appli biochar . The eme biochar .+ transport channels are overtaken by Na+ leading to lower and reduced plant growth [+ uptake and improving K+/Na+ ratio. For instance, co-application of either of the two endophytic PGPRs, Burkholderia phytofirmans or Enterobacter sp, with biochar significantly mitigated the salinity stress in maize by reducing the xylem Na + uptake [Pseudomonas koreensis and Bacillus coagulans PGPRs with biochar significantly increased the K+ and K+/Na+ ratio thereby leading to lowered salinity stress in rice plants [+ in soil solution were also decreased by the latter’s addition to adsorption sites and desorption of K+ by co-application of PGPRs and biochar. Another PGPR Burkholderia phytofirmans, which is capable of producing exopolysaccharides, when inoculated along with biochar significantly, decreased salinity stress for plants by lowering Na+ content in soil solution. In addition to lowering Na+ content, co-applying PGPRs with biochar enhances colonization efficiency of the former thereby leading to synergistic effects on soil quality. For instance, Akhtar et al. [Burkholderia phytofirmans and Enterobacter sp. strains co-applied with 5% biochar (derived from hard wood and soft wood) in a saline soil than PGPRs without biochar in soil. Enterobacter sp with 5% biochar showed high colonizing efficiency in saline soil than Burkholderia phytofirmans with and without 5% biochar. Similarly, co-application of an endophytic PGPR with biochar to Chenopodium quinoa grown in a saline soil induced an increase of  ~ 150–250% in PGPR colonization in rhizosphere, root interior and shoot interior bacterial population than PGPR inoculation alone. In presence of biochar studies showed a decreased Na+/K+ ratio in soil and increased root colonizing efficiency of PGPRs hence alleviating salinity stress in soil. In soil solution, biochar and PGPRs maintain the nutrient balance by releasing mineral nutrients such K+, Ca2+ and Mg2+, thereby reducing Na+ in soil. This ultimately increased the K+/Na+ ratio in soil. Exopolysaccharide produced from PGPRs under stress binds Na+ in soil [As far as the soil quality is concerned, salinity reduces microbial activity and biomass in addition to changing the microbial community structure in soil . Moreovet growth . However+ uptake . Similare plants . In the r et al.  reported in soil .Enterobacter sp. (PGPR) inoculums with biochar (paper and pulp derived) could be an efficient approach to accelerate remediation of soil contaminated with cadmium (Cd) (80 mg kg−1 soil). Although PGPR and biochar immobilized Cd in soil thereby mitigating its availability by 15.2% and 28.3%, respectively, their combination decreased it by 45.6%. Another PGPR, Bacillus sp. in the presence of biochar increased soil enzyme (dehydrogenase) 4.61 times high than biochar leading to increased bioremediation. This combination also decreased HOAc-extractable Cd level by 11.34% than sole applications of biochar or PGPR [Bacillus sp with 1% biochar significantly reduced the toxic effect of chromium and improved plant health by limiting the availability of the heavy metal [The use of PGPR in combination with biochar has also been studied in polluted soils Table . On the  or PGPR . The appvy metal . Both PGvy metal .Achromobacter xylosoxidans, Pseudomonas aeruginosa, Leclercia adecarboxylata, and Enterobacter cloacae with timber waste biochar (0.75 and 1.50% w/w) in drought conditions improved the growth of maize by inducing higher nutrients uptake and lower ethylene level than sole application of biochar or PGPR [A. xylosoxidans + 1.50% biochar showed 19 and 6% higher transpiration rate, 30 and 7% higher photosynthetic rate, and 16% and 7% higher stomatal conductance, respectively, than alone A. xylosoxidans or 1.5% biochar under severe drought. E cloacae + 1.5% biochar increased chlorophyll a by 26 and 13%, carotenoids by 28 and 4%, and total chlorophyll by 29 and 9%, respectively, than E. cloacae or 1.5% biochar, respectively. Similarly combined application of P. aeruginosa and biochar decreased electrolyte leakage by 28% and 4% than applying P. aeruginosa or biochar alone, respectively. Similarly, Nafees et al. [Cellulomonas pakistanensis and Sphingobacterium pakistanensis PGPRs and biochar derived from wood of Morus alba (5% w/w) on Vicia faba under drought stress. They found that co-application positively ameliorated fresh and dry leaf weight by 28.57 and 10.47%, fresh and dry root weight by 36.36 and 14.28%, and fresh and dry shoot weight by 16 and 10% than sole application of biochar or PGPR, respectively. Some other ACC deaminase producing PGPRs, i.e., Agrobacterium fabrum and Bacillus amyloliquefaciens have also been found to boost wheat productivity under severe drought when used in combination with timber waste biochar [B. amyloliquefaciens + biochar increased plant height by 34 and 24%, root length by 25 and 8%, and spike length by 5 and 2% than B. amyloliquefaciens or biochar alone. Similarly, A. fabrum + biochar increased 1000-grains weight by 13% when compared to sole application of A. fabrum. Ullah et al. [Serratia odorifera, and algal biochar on maize growth under drought stress. The co-application increased maize growth parameters like plant height by 38 and 16%, shoot fresh weight by 29 and 17%, shoot dry weight by 44 and 24%, root fresh weight by 60 and 27%, root dry weight by 84% and 24%, and root length by 47 and 32% than sole application of PGPR or biochar under severe drought stress, respectively. Decreased proline content due to combined application of PGPRs and biochar has also been cited as drought alleviating mechanism [Pseudomonas koreensis and Bacillus coagulans, when used with biochars, on rice plant under drought conditions increased relative water content, stomatal conductance, Ca2+ and K+ content and decreased proline content in plants. Another PGPR, P. fluorescens, when applied along with biochar to cucumber under limited moisture conditions was found in much higher number than when it was applied alone [Many studies have reported the effect of combined application of PGPR and biochar on plant productivity under different environmental stressors Table . They ha or PGPR . Brieflys et al.  investig biochar . B. amylh et al.  evaluateed alone . Their cBurkholderia phytofirmans in combination with tree-twig derived biochar improved plant height, root dry weight, shoot dry weight, grain yield, photosynthetic rate, stomatal conductance of Chenopodium quinoa by 17, 26, 10, 5, 5, 16 and 12%, respectively, under saline conditions than individual PGPR application only [Bacillus coagulans and Pseudomonas koreensis were co-applied with rice husk-derived and corn stalk-derived biochars in a rice field having electrical conductivity of 4.67 dS m−1 biochar. The co-application alleviated the negative effects of salinity by decreasing Na+ content by 15.34% and 15.73%, and proline content by 52.49% and 49.57% in first and second year of the study, respectively, in rice leaves, in comparison to the uninoculated control [+ uptake than biochar or PGPR sole applications, respectively, by using Enterobacter with 5% biochar and Burkholderia phytofirmans with 5% biochar derived from hard and soft wood in saline soil.Soil salinity affects plant growth, development and photosynthesis. It also affects protein synthesis and lipid metabolism . Plant gion only . Evidenc control . Similar control  found 25Alcaligenes faecalis and Bacillus amyloliquefaciens and provided with compost (mixed fruits) mixed biochar (vegetable waste). Resultantly, they found that A. faecalis strain along with compost-mixed biochar significantly improved plant chlorophyll content by 37%, root dry weight by 58%), nitrogen by 46%, phosphorus by 39%, and potassium by 63% in mint leaves than untreated control. In another study, the lead uptake in spinach decreased by 43% whereas potassium uptake increased by 10.5% over untreated control by the use of compost-mixed biochar and Bacillus amyloliquefaciens strain [Enterobacter sp. when co-applied along with biochar significantly enhanced growth of Brassica napus in cadmium-spiked (80 mg kg−1) soil [−1) than biochar only (0.42 g pot−1) and lowest Cd concentration (5.45 mg kg−1) was found in PGPR + biochar treatment as compared to biochar, PGPR and control (soil without PGPR and biochar).PGPR and biochar play an important role in the management of heavy metal stress in plants. They can transform, accumulate or detoxify heavy metals . For inss strain . The PGP−1) soil . The co-−1) soil  found anThe synthesis of literature so far in this paper has amply highlighted that the biochar and PGPRs work synergistically in improving the soil quality and agriculture productivity. When biochar is applied with PGPR inoculants, it provides habitat for PGPR  due to its porous structure and high surface area and also the ability to adsorb microorganisms and organic compounds . Some stPaenibacillus polymyxa, Rahnella sp., Serratia sp. have the ability to enhance the mineral nitrogen content in soil solution through their nitrogen-fixing traits and prevents its leaching in soil [Bacillus sp., Bacillus lentus, B. subtilis, Bacillus megaterium, Burkholderia sp., Glomus etunicatum, G. mosseae, Pseudomonas species, Pseudomonas fluorescencs Penicillium strains, Lysinibacillus fusiformis, Azotobacter chroococcum, Azospirillum brasilense, Arthrobacter, Streptomyces, have been shown to solubilize and provide phosphate in soil for plant uptake [Enterobacter sp., Alcaligenes sp., Pseudomonas fluorescens, Serratia odorifera. Leclercia adecarboxylata, Agrobacterium fabrum, Bacillus amyloliquefaciens, Pseudomonas aeruginosa, etc., have the ability to produce ACC deaminase. These strains show synergistic effects with biochar in abiotic stress alleviation [PGPRs are involved in plant growth promotion under normal and stressed conditions through their direct and indirect mechanisms. Similar to biochar, the PGPR may either bring in a nutrient from outside through their direct mechanism such as nitrogen fixation (by nitrogen-fixing bacteria) or solubilize the immobilized nutrients (by phosphate-solubilizing bacteria) thereby contributing to plant nutrition. For instance, nitrogen-fixing PGPRs such as  in soil , 117. A t uptake . While tt uptake , 96. Anot uptake . PGPRs seviation , 80, 104eviation .13C, 15 N, 33P) in order to trace the carbon and nutrients accrued into microbial biomass. Concurrently, the colonization efficiency of the PGPR should also been estimated.Mechanistic understanding of the interaction between PGPR and biochar needs further exploration. For instance, currently we don’t know exactly if the synergistic effect of the two is because of the conducive habitat afforded to the PGPR by biochar or it is due to the enhanced availability of substrate and nutrients due to biochar that sustains and promotes the PGPR. It can be done by using isotopically labeled biochar (i.e., Long-term field experiments could be a highly effective way of evaluating the combined effect of the PGPR and biochar. Individually, the PGPR and biochar have been assessed in reasonably long-term experiments for their potential for sustainable food production –14, 121.The PGPR technology is not very successful in degraded soils situated in semi-arid and arid areas, especially which are poor in soil organic matter, because the PGPR have not good reserves of substrate and nutrient-source for their growth and function. Combined application of the PGPR and biochar in these soils could be a very good strategy and needs to be assessed. The biochar may provide the PGPR the habitat to survive and flourish as well as the necessary substrates, which are lacking in such soils, and is the key reason of failure of PGPR technology there.Meta-analyses of the studies on biochar vis-à-vis agricultural productivity have revealed that the major mechanism by which they improve productivity is the liming effect , 82. SucUnder different environmental stresses, low crop growth and crop failure is the norm across many important food and cash crops. Co-application of PGPR and biochar offers a sustainable, cost-effective, and environment-friendly technique for increasing crop productivity and improving soil quality. Even under normal conditions, this combination may act synergistically to improve crop productivity as well as soil quality in addition to lowering the need for chemical fertilizers. However, as is highlighted by this review, there are not many field experiments that have been conducted to explore the potential of combined application of the PGPR and biochar for sustainable food production. Given the state-of-the-art of the subject, we have following recommendations for future studies:


The emergence of point-of-care (POC) testing has lately been promoted to deliver rapid, reliable medical tests in critical life-threatening situations, especially in resource-limited settings. Recently, POC tests have witnessed further advances due to the technological revolution in smartphones. Smartphones are integrated as reliable readers to the POC results to improve their quantitative detection. This has enabled the use of more complex medical tests by the patient him/herself at home without the need for professional staff and sophisticated equipment. Cytokines, the important immune system biomarkers, are still measured today using the time-consuming Enzyme-Linked Immunosorbent Assay (ELISA), which can only be performed in specially equipped laboratories. Therefore, in this study, we investigate the current development of POC technologies suitable for the home testing of cytokines by conducting a PRISMA literature review. Then, we classify the collected technologies as inexpensive and expensive depending on whether the cytokines can be measured easily at home or not. Additionally, we propose a machine learning-based solution to even increase the efficiency of the cytokine measurement by leveraging the cytokines that can be inexpensively measured to predict the values of the expensive ones. In total, we identify 12 POCs for cytokine quantification. We find that Interleukin 1 Point of care (POC) tests also play a significant role in combating the COVID-19 virus, as any person can test him or herself at home for the presence of an infection with the virus within a few minutes. As the above examples show, POC tests are used for various purposes, including monitoring therapy, managing a disease or maintaining the health of a patient. Thereby, the patient is given more responsibility but also more control over him or herself.In developing POC tests, researchers have also recognized the potential of recent technology developments such as smartphones and machine learning e.g., ,2). This. This2])Therefore, it is necessary to identify the state of development of cytokine tests suitable for home usage by the patient him/herself, especially those that require little or already at-home existing equipment—such as a smartphone.1–RQ3). The distinction between expensive and inexpensive is made based on how easy it is to acquire the cytokines with the technologies identified. In other words, the detection of cytokines is categorized as inexpensive if the technology used to measure the cytokines is mature and uses equipment that is available in most households, such as a smartphone or a sheet of paper. Otherwise, the technology is categorized as expensive.Furthermore, machine learning algorithms have been widely used in the medical domain. A machine learning model is used to predict the levels of inflammatory cytokines in patients with Parkinson’s disease . Wei et 1: What recent technologies developed for the rapid determination of cytokines are suitable for at-home testing?RQ2: Which technologies suitable for at-home testing can be used to acquire cytokines inexpensively and which expensively and which cytokines do these technologies measure?RQ3: To what extent can cytokines measured by inexpensive technologies be used to predict levels of cytokines that can currently be tested only by expensive technologies based on the public dataset 10k Immunomes?RQThis study aims at answering three research questions:1.We use the PRISMA method to collect POC technologies that can be used for at-home testing with a focus on methods that leverage smartphones for cytokine quantification to answer RQ2, we group the retrieved POC technologies into expensive and inexpensive cytokines based on the equipment and the personnel required to conduct the test.To answer RQ3 by building machine learning models that take as input the inexpensive cytokines and predict each of the expensive ones. Then, we evaluate the performance of the models to validate the potential of delivering reliable predictions for each of the expensive cytokines. Thus, the quantification of expensive cytokines can be carried out without the need for any expensive test.We answer RQIn this study, our main contributions can be described as follows:1 and RQ2 in 3. The regression analysis results are presented in This work is structured as follows: in In 2021, we published a review  on the sLiu et al. summarized on account of the COVID-19 pandemic in their 2021 review [Hernández-Neuta et al. investigated in their 2019 review . AnotherHernández-Neuta et al. indicate that the biosensors found are mostly in a proof-of-concept phase and only a few are in the product development phase. The biosensors must still be validated and prove their feasibility in the clinical field .Romeo et al. investigated in their 2016 review  the receod serum .Already in 2015, Chen et al. discussed in their review [Mechanical cytokine biosensors “detect surface binding events of biological molecules by means of mechanical deflections or dynamic resonance shifts of the transducer system resulting from surface stress or mass changes”  (p. 9). Optical biosensors function in such a way that optical signatures are investigated that arise from the refractive index changes of a solid light-guiding microstructure  upon the surface binding of analytes . RepreseFurther reviews focus on giving an overview of the advances of biosensors at the POC for the detection of cytokines but also other biomarkers  or the In summary, we find that some of the reviews either do not focus on the at-home testing or address POC technologies for measuring disease-related biomarkers other than cytokines. In this manuscript, we focus on collecting POC technologies for measuring cytokines at home, possibly with the help of a smartphone, then divide them into technologies that can acquire cytokines inexpensively and those that can acquire them but expensively.Indeed, cytokines are vital proteins that play a major role in the human body. Therefore, numerous studies have analyzed the correlations between each other and other disease and health conditions. Vculic et al. investigate the correlations among cytokines and age in healthy subjects and among cytokines and age in patients with autoimmune thrombocytopenic , whereasFurthermore, due to the recent popularity of machine-learning methods, especially in biology and medicine, various studies present machine-learning methods that exploit the measured cytokines to predict the progression of a disease ,27 or to1) and two (RQ2), a systematic literature review is conducted according to PRISMA. PRISMA is the abbreviation for Preferred Reporting Items for Systematic Reviews and Meta-Analyses [In order to answer the research questions one Analyses . The maiAnalyses  (p. 180)Analyses .For this work, the PRISMA items, eligibility criteria (item 5), information sources (item 6), search strategy (item 7) and study selection (item 16) will be applied. The following is a brief explanation of what the selected PRISMA items contain. 1) has to be already applied in this step, as the syntax for the search in the title and abstract must be adapted accordingly. The identified reports are then assessed for eligibility according to the inclusion and exclusion criteria  presented in 2) is based on the characteristics for home tests found by Jamaludeen et al. in their review [5) for other unprocessed samples such as saliva, sweat or urine. Inclusion criteria three (IC3) shows that the review focuses on cytokines. In addition, the technologies identified have to be mature in terms that these are either already in place or tested prototypes already exist (IC4). With exclusion criteria one (EC1), a year limit for scientific publications not older than 2015 will be applied due to technological developments in recent years . Studies that report on technologies that are limited to specific conditions  will be excluded because of exclusion criteria two (EC2) as the review intends to find technologies for a general application. Exclusion criteria three (EC3) limits the results for German and English languages.First, the search queries are applied to a total of five databases using the specific search syntax for each database. Inclusion criteria one , the literature review results are listed. In the overview, all technologies found will be named with the measurable cytokines, the technique behind it, the sample type and, if possible, the sample quantity and sample time. Further, the overview is the basis for answering research question two (RQ2), in which the technologies found are to be grouped according to whether the cytokines can be acquired expensively or inexpensively. The results are displayed in two tables: one for cytokines that are expensive to acquire and one table with cytokines that are inexpensive to acquire, each of which also lists the cytokines that are measured with them. The cytokines found, which can be acquired inexpensively through the identified technologies, are then used as independent variables for implementing several regression models for answering the remaining research questions .To answer research question one (RQ1–RQ4).1 was applied (n = 6)  stated in the introduction were used to conduct the PRISMA literature review.The search queries (SQ3) of the eligibility criteria.In the PubMed and IEEE Xplore databases, a search of abstract and title was possible as intended.In the Scopus and ScienceDirect databases keywords were searched in addition to the abstract and the title, due to the given limitations of search parameters by the databases mentioned.1) could not be fully applied by search parameters. It was possible to conduct a title search but this did not return any hits. An abstract search was not possible. Therefore, a full-text search was conducted but for each search query, there were several thousand hits that could not all be evaluated. Google Scholar is a well-known and well-used database and encompasses all relevant databases, including the other used databases in this research. Therefore, the database was not excluded; instead, every search query was limited to the first 10 result pages which equal 100 hits per search query. Afterward IC1 was applied manually. The full-text search seemed to be a good addition to the search in the title and abstract.The search on Google Scholar was slightly different from the other databases as inclusion criteria 1 (IC1):1: Does the title or abstract contain one of the search terms (SQ1–SQ6)?ICFor the identification of suitable papers, inclusion criteria 1 was applied first . A total of 48 records were duplicates and therefore removed as well while screening the records. In the end, a total of 180 reports were assessed for eligibility. Most of them (n = 166) failed the eligibility criteria . One remark is that none of the retrieved studies reported their results in German. Two more reports were excluded because the tests were limited to animals. Finally, 12 reports remained on technologies that can be used to measure cytokines at home. Not listed among the included reports in the flow diagram are six reports that mention technologies that do not measure cytokines but do measure biomarkers related to the immune system. Therefore, these reports failed IC3.1) and two (RQ2):1: What recent technologies developed for the rapid determination of cytokines are suitable for at-home testing?RQ2: Which technologies suitable for at-home testing can be used to acquire cytokines inexpensively and which expensively and which cytokines do these technologies measure?RQThe findings of the PRISMA literature review make it now possible to answer the research questions one  iAlso for the aptamer-based biosensor from Wang et al., the sample time is not clearly stated . They saentioned . This deWang et al. developed another GFET biosensor that was manufactured on a bio-compatible and ultra-thin, polymer-supporting substrate  in 2020 vailable .The QCM biosensor  of Pohanod serum . The limod serum  (p. 4) aod serum  (p. 4). The paper biosensor  of Adrovnd IL-8”  (p. 1). nd IL-8” . The repPunj et al. developed a self-assembled monolayer (SAM) multiplex electrochemical biosensor  that meaAlba-Patiño et al. developed in 2020 a plasmonic-based mobile biosensor  made of Another electrochemical biosensor that is based on an impedance spectroscopy (EIS) system  was deveedicine”   strips” . The rea strips” . The dev strips” .A hybrid magneto-electrochemical sensor  was deveand IL-6 .2, In order to answer RQInterleukin 1sponses”  (p. 922)sponses” . Interlee storm”  (p. 5). e storm” . Furthere storm”  (p. 5). e storm” . Tumor n process . TNF-α iammation . Interfeections”  (p. 1).C-Reactive Protein (CRP), Prostate Specific Antigen (PSA) as well as both antibodies Immunoglobulin G (IgG) and ImmunoglobulinM (IgM) have also been observed during the literature review and are listed in Bouraoui et al. were able to identify an association between elevated proinflammatory cytokines (TNF-e cancer . The MCFe cancer  (p. 1).Choi et al. developed the Eigen Diagnosis Platform (EDP) as “a portable quantitative immunoassay platform”  and demoWe use a public dataset of healthy human subjects collected for analyzing the immunological mechanisms in the immune system  to answeTo answer the third research question, the cytokines summarized in The development of the regression models is implemented with the programming language Python.Three regression algorithms with their different advantages and disadvantages were chosen for the training of regression models based on the public dataset 10k Immunomes: (multiple) linear regression, decision tree and random forest.Multiple linear regression concentrates on the linear relationship between the dependent target variable and more than one independent input variable . TherefoThe (multiple) linear regression algorithm is easy to understand and to implement but it also has some downsides . Linear In contrast, the second selected regression algorithm is the decision tree algorithm which is an example of a non-linear method.The biggest advantage of the decision tree algorithm is that it can handle next to non-linear data and also linear data and compared to other algorithms a decision tree is quick to train . FurtherA possibility to avoid overfitting is to use the random forest model. Here, various decision trees are generated to combine the predictions of them .The random forest algorithm generates randomly several training datasets from the original dataset by using the bootstrapping method . The metAll three models presented are very different in their approach. They have different strengths and weaknesses, with some weaknesses of one algorithm being mitigated by the approach of another of the selected algorithms . It is to be expected that the selected regression algorithms will perform differently and produce different “good” models. With this in mind, a comparison of these models seems very interesting.It is important to conduct pre-processing of the data before building the regression models. In , Manasa Another essential step in preparing the data for a later model is feature selection. Since the aim of this work is to find out whether the identified features from the results of research questions one  . The hypThe aim of the implementation of different regression models was to answer research question three  and are still in the prototype phase. Additionally, further technologies could be identified that measure no cytokines but other immune-related biomarkers. The identified technologies were divided into inexpensive and expensive technologies depending on whether the cytokines are easy and inexpensive to acquire with these technologies or not. Inexpensive-to-acquire cytokines suitable for home testing could be identified: Interleukin 1The PRISMA literature review results were the basis for answering research question three (RQThis study focuses on modeling the dependencies between the expensive and the inexpensive cytokines in healthy subjects. However, it is also necessary to analyse these dependencies in subjects with some health conditions because we expect these dependencies to differ when the studied subjects have a disease.Quantifying the cytokines that play vital roles in the immune system allows for a reliable assessment of the immune system’s fitness and the body’s health. Therefore, developing an efficient method that does not require many resources is necessary. We believe that exploiting the inexpensive cytokines to quantify the expensive ones moves us one step closer to assessing the fitness of the immune system without the need for complex equipment and professional staff.


A total of 601 unique species were detected across all samples. Alpha diversity richness  and evenness/dominance  metrics revealed no significant differences by birth mode. Beta diversity , as estimated with Bray‐Curtis dissimilarities and Aitchison distances (using log[x + 1]‐transformed counts), was also not significantly different . Only the abundance of Lachnoclostridium [Clostridium] scindens was found to differ using a combination of statistical methods , being significantly higher in C‐section mice. This microbe has been implicated in secondary bile acid production and regulation of glucocorticoid metabolism to androgens. From our results and the extant literature we conclude that C‐section does not inherently lead to large‐scale shifts in gut microbiota populations, but birth mode could modulate select bacteria in a context‐specific manner: For example, involving factors associated with pre‐, peri‐, and postpartum environments, diet or host genetics.In humans and animal models, Cesarean section (C‐section) has been associated with alterations in the taxonomic structure of the gut microbiome. These changes in microbiota populations are hypothesized to impact immune, metabolic, and behavioral/neurologic systems and others. It is not clear if birth mode inherently changes the microbiome, or if C‐section effects are context‐specific and involve interactions with environmental and other factors. To address this and control for potential confounders, cecal microbiota from ~3 week old mice born by C‐section  Lachnoclostridium [Clostridium] scindens was significantly higher in C‐Section mice. (B) Leptotrichia trevisanii abundances differed by birth mode, due to a sub‐set of natural birth animals positive for the microbe. Sample sizes are n=23  and n=16 . Thus, the field awaits a definitive answer to the question, “Does birth mode, by definition, inherently lead to a difference in the gut microbiome?” Considering the potential significance of this to public health and clinical practice, it is imperative to answer this question through studies designed to examine the effects of birth mode while minimizing confounders. To this end, we conducted a controlled mouse model experiment to observe whether C‐section affects the cecal microbiota when compared to offspring derived from vaginal birth. We reasoned that if birth mode inherently leads to significant differences in cecal microbiota, this would be detectable in young animals tested at the ~3 weeks postpartum, peri‐weaning stage.22.1n = 3) were anesthetized for C‐section with isoflurane (oxygen flow 1.0 LPM and mix of 2.5%–3.5%). For this protocol, the dam was laid on its back, the abdomen shaved and cleaned with alcohol and betadine. An incision was made the full length of the abdomen to gain access to the peritoneal cavity. The uterine horn was pulled out and laid on a warmed saline‐moistened gauze pack. The horn was cut along the whole length with scissors, along the side opposite of the placenta. Pups were pulled away from their connections with the uterus so the complete fetus with amniotic sac and placenta were removed. The pups were removed from their amniotic sacs and gently wiped clean and dried with warmed gauze. Pups were then placed on a heated pad  until dry, warm, pink and beginning to move. Pups were then transferred to a CD‐1 foster dam after her entire litter had just been removed. C‐section offspring were weighed two consecutive postnatal days  to confirm positive weight gain. The offspring from natural delivery dams (n = 4) offspring were cross‐fostered approximately 1 day after parturition (placed with recipient dam at ~10:00 the day after parturition) and weighed on PND2. C‐section dams cross‐fostered 14 males/18 females to three EMD7 dams, with a total of four deaths during PND1. Natural‐birth dams cross‐fostered 25 males/19 females to four EMD7 dams, with no deaths. Typical cross‐fostered litters were 11 or 12 pups, except one cross‐fostered C‐section litter had 10 (due to a single pup PND1 death) and one cross‐fostered natural born litter had 17.Studies were approved by the UAMS Animal Use Committee (Protocol #3740). Timed pregnant CD‐1 IGS mice (strain code 022) were supplied by Charles River. First‐time pregnant mothers 8–10 weeks of age were timed bred to arrive on embryonic day 4 and day 7 . The dams were single‐housed in cages with TEK‐Fresh laboratory animal bedding (Envigo‐Teklad 7099) and a single nestlet , and fed ad libitum rodent chow (Envigo‐Teklad 8640) and water. The vivarium light cycle was 12 h:12 h, and the housing temperature 22°C. EMD7 dams served as foster dams and had offspring at gestation day 19. EMD4 dams served as the source for naturally‐born and C‐section offspring. On gestation day 19, a subset of EMD4 dams  following the manufacturer's instructions. For each sample, total DNA concentration, purity, and quality were measured using the AATI Fragment Analyzer (Agilent). Extracted DNA was stored at −80°C until library preparation. DNA libraries were prepared using the Nextera DNA Library Prep kit (Illumina) followed by shotgun sequencing performed on an Illumina NextSeq 500 platfrom with a 2 × 150‐bp, paired‐end run. Raw sequence data files were demultiplexed and converted into FASTQ files using Casava v.1.8.2 (Illumina). MEGAN Ultimate Edition software (v6.18) was used to analyze the reads by performing taxonomic binning and assigning reads to respective nodes in the National Center for Biotechnology Information (NCBI) taxonomy  using log (x + 1)‐transformed counts, visualized with Principal Co‐ordinate Analysis (PCoA). Birth mode differences in beta‐diversity was determined with Permutational Multivariate Analysis of Variance (PERMANOVA). Analyses of bacterial species differences were performed using the ALDEx2 v1.24  < 0.05. All statistical analyses of microbial sequencing data were conducted in the R Statistical Language (v 4.1.0). Sequencing files are publicly available on the National Center for Biotechnology Information Sequence Read Archive repository with the accession number PRJNA841368.Alpha‐diversity measures were calculated using the estimated richness . In a subset of mice (used in separate studies) measured at PND 7/8, this relative difference remained in natural‐born versus C‐section pups . However, by PND 21/22, in the sub‐group of mice used for metagenome analyses herein, body weights no longer differed .At PND2, average body weight was slightly (~12%) but significantly higher in natural‐born compared to C‐section cross‐fostered pups .There were 39 samples with 16 total mice born via C‐section and 23 from natural vaginal birth. Of those mice, 19 were female and 20 were male. A total of 601 unique species were observed across all samples. All results for statistical analyses are provided in Table p = 0.094) and Aitchinson distances (p = 0.089).Alpha diversity metrics of richness and evenness are presented in Figure Lachnoclostridium [Clostridium] scindens was significantly higher in C‐section mice versus natural birth mice . Environmental confounders were further minimized by examining the microbiome at an early age, just prior to weaning. The results indicated that few differences in microbiota populations were apparent at PND 21/22, highlighting that birth mode alone may not fully explain reported effects of C‐section on offspring gut microbiome. It is acknowledged that this perspective stems from a single age analysis, and we cannot discount the possibility that birth mode impacts microbiome more profoundly at different life stages.Bifidobacterium spp. in male C‐section NIH Swiss mice (at 3 weeks) was detected, and there were also early‐life social, cognitive and anxiety differences  and a lower abundance of Candidatus arthromitus  in C‐section mice. The investigators speculated that differences in the gut microbiome measured at age 5 weeks may have contributed to behavioral phenotypes. Aspects that could make study‐to‐study comparisons challenging are the differing types of biospecimens , analysis methods , feeding regimens, and ages that can be employed across experiments.The largely “null” result in the current study with respect to C‐section shifts in gut microbiota contrasts with several papers reporting birth mode microbiota differences in mouse models. For example, behavioral changes in C‐section mice have been hypothesized to be linked to differences in specific gut microbiota. In one study, lower relative abundance of fecal IL‐10 in lymph nodes and spleen. While no birth mode‐associated differences were seen in adult microbiota, the C‐section mice around weaning had significantly lower relative abundances of Rikenellaceae and Ruminococcus plus higher Bacteroides acidifaciens and Lachnospiraceae in feces when compared to vaginal birth mice   that was significantly increased in cecum from C‐section offspring. This species is an archetypal bile acid modifying microbe that expresses the enzyme 7α‐hydroxylase that drives conversion of cholic acid to deoxycholic acid  and 7β‐hydroxylase that catalyzes conversion of DCA to 7‐epicholic acid . Bacteremia associated with this “opportunistic” microbe has been reported and seems to manifest primarily in the immunocompromised condition . Additional support was from USDA‐ARS Project 6026–51000‐010‐05S.Sean H. Adams is the founder and principal of XenoMed, LLC, which is focused on research and discovery in the area of microbial metabolism. XenoMed had no part in the research design, funding, results or writing of the manuscript.Table S1Click here for additional data file.


One of the indigenous sheep breeds that is primarily produced in Kenya under an extensive production system is the Red Maasai sheep. It has a reputation for being able to thrive in arid and semi-arid environments. A common belief is that, when contrasted with the Dorper breed, it performs poorly in terms of growth traits. Therefore, to enhance their growth traits, the Red Maasai are randomly crossed with Dorper. In contrast, the Red Maasai sheep breed outperformed the Dorper sheep breed in this study in terms of post-weaning growth rate, once more demonstrating early resilience and adaptation to local environmental conditions independent of maternal influence.p < 0.05) influenced birth weight; breed, sex, and birth type all significantly (p < 0.05) influenced weaning weight, whereas season, sex, and dam age significantly (p < 0.05) influenced yearling weight. The RedM breed outperformed the Dorper breed in post-weaning growth rate, demonstrating early resilience and adaptation to local environmental factors independent of maternal influence. Breed and non-genetic factors play a vital role in the growth of lambs, and the results of this study offer an opportunity for improved farm management under an extensive production system and selection for the conservation of the indigenous Red Maasai breed.This comparative study aimed to evaluate genetic and environmental factors’ effects on the growth traits of lambs in Dorper and Red Maasai (RedM) sheep breeds. The data analyzed contained the following measurements: birth weight (Bwt), weaning weight (Wwt), yearling weight (Ywt), birth type (single or twins), and details on each lamb’s dam (dam ID and age) and sire. Except for the RedM, whose birth weight decreased with time, both breeds generally showed an increase in other growth weights across the study period, with yearly variations affecting both breeds. Additionally, analysis by a linear mixed model with restricted maximum likelihood (REML) showed that only breed as a factor significantly ( Small ruminants in Kenya contribute significantly to the Kenyan national economy, with a value of USD 688 million USD in 2020, up from USD 469 million in 2016 [More than 60% of the sheep populations in Kenya are native and predominantly kept under an extensive farming system, majorly by pastoralists and resource-poor farmers ,4. TheirHaemonchus contortus [In Kenya, sheep breeds are mainly classified as either fat-tailed, which includes the Red Maasai (RedM) and East African fat-tailed breeds , or fat-ontortus , its tolontortus , and itsThe Dorper sheep breed, which originated from South Africa, is a hair meat sheep ,9 and a As a result, the Kenyan government established sheep and goat farms in some parts of the country in different ecological zones to preserve the most vulnerable indigenous small ruminant breeds, particularly the RedM sheep breed. The main mandate of these farms is to objectively select and breed pure RedM to sell breeding stock/rams to farmers to upgrade their stocks. Alongside RedM, Dorper sheep are also bred, and farmers use Dorper rams to crossbreed with local RedM for the improved growth performance and meat traits of the progeny. In this case, Dorper rams are used as terminal sires, with the assumption that all the lambs are slaughtered to control native gene erosion.To our knowledge, this is the first study aiming to establish the phenotypic trends and the correlation between quantitative phenotypes as well as examine the breed and non-genetic factors affecting the growth performance of the Dorper and the indigenous RedM breeds kept at the Naivasha sheep and goats station farm in Kenya. The results of this study will provide an opportunity for improved flock management under an extensive management system for an improved in situ indigenous sheep conservation plan in Kenya.Data used in the present study were obtained from the Naivasha sheep and goat station (Ol’magogo farm) located at latitude −0.551494 and longitude 36.413791. The station is managed by the government of Kenya under the Ministry of Agriculture, Livestock, Fisheries and Cooperatives within the State Department of Livestock Development. To improve the productive potential of the indigenous sheep breeds, particularly RedM, the government of Kenya, in collaboration with the Food and Agricultural Organization (FAO), initiated a genetic improvement program in the 1970s with a focus on within-breed selection and crossbreeding between the RedM and Dorper  breeds tCynodon plectostachyus) with scattered tall acacia trees (Acacia xanthophyloea). The soil is volcanic in origin, alkaline (pH 7.4), sodic and deep.Ol’Magogo is in Kenyan ecological zone IV, which is classified as semi-arid. Historically, the region experiences a bimodal rainfall pattern, with March–May being the long-wet period, while October–December is the short-wet period. On average, the region receives a poorly distributed rainfall of 680 mm per annum. The average minimum and maximum temperatures range between 8 °C and 28 °C, respectively, with a relative humidity of between 60% and 70%. The elevation of this region ranges between 1830 and 2330 m a.s.l. (meters above sea level). The natural vegetation is predominantly star grass . Generally, ewes weighing below 25 kg were not joined. The farm has two mating seasons: October–November and April–May, which leads to lambing in August–September and February–March, respectively. The age at first joining was 12 to 18 months, and longevity depended on the reproductive performance of the animal. Lambs were left to run freely with their mothers until weaning (90 to 120 days of age). Feed supplementation was seldom done, even in nursing dams. However, mineral-licking blocks were provided. Water was also provided ad libitum. Disease and parasite management measures were mainly prophylactic and biosecurity oriented. Animals were routinely weighed at birth, weaning, yearling and before breeding. Flushing by grazing ewes on regenerative pasture was done one month before breeding season to improve the breeding flock’s body condition score, as indicated in ,14.The Ol’magogo Sheep and Goat Station in Naivasha, Kenya provided the 903 growth performance records for the Dorper and RedM sheep breeds, which were evaluated for the years 2007 through 2020. Data collection was done as part of station record-keeping procedures, not for the goal of this study. The information on growth performance includes the lambs’ birth weight (Bwt) at birth, weaning weight (Wwt) on the 90th day after birth, and yearling weight (Ywt) on the 365th day after birth. The average daily gains (ADG1) and (ADG2) before and after weaning, respectively, were subsequently determined. Additionally, each lamb’s sire and dam were matched. The dam’s age and her tupping weight (Pwt) were also included. Phenotypic trends of lambs’ weights  over the years between 2007 and 2020 were plotted using the ggplot2 package. Further average daily gain pre-weaning (90 days post lambing) and post-weaning (275 days after weaning) were compared between both breeds using the Wilcoxon test  implemenThe linear model  procedurThe phenotypic trends for each growth trait under investigation were analyzed and are presented in p < 0.05) influenced weaning weight. On the other hand, the yearling weight was significantly (p < 0.05) influenced by the season, sex, and the dam’s age. Dam age was regarded as a continuous variable in this instance. The mean yearling weight increased with an increase in dam age, as presented in p < 0.05) heavier than females, and singletons were significantly (p < 0.05) heavier than twins at weaning. Dorper lamps were significantly (p < 0.05) heavier than RedM lambs at birth (p < 0.05) heavier.A summary of the fixed effects, their significance, and their corresponding residual standard deviations are shown in at birth , and theHaemonchus contortus [In general, both breeds showed an increase in all growth weights, with yearly variances affecting both breeds , except ontortus ,29,30,31ontortus  than Dorontortus  recommenSurprisingly, only breed had a significant effect on birth weight among all the supposedly fixed-effect components. Dorper lambs were significantly heavier than the RedM lambs by 0.58 kg. This could be due to the larger mature size of the Dorper breed ,7,32. ThThe sex of lambs also significantly influenced the variation of Wwt and Ywt. Males were generally heavier than females at weaning and the first 365 days of life. This indicates the existence of sexual dimorphism in both breeds, and these results are comparable to the previous findings such as ,35,36. Sp < 0.05) heavier than lambs born in the September/October lambing season. However, the season has no influence on lamb Bwt and Wwt. The long wet season, which lasts until August in the study area, coincides with the lambing season in April/May. The sheep flock would be able to meet their nutritional needs, as there is an abundance of fresh foliage during this time. Lambs would consequently grow more quickly. The long wet season is immediately followed by the short wet season (October-December), which is then followed by a dry period (December-February), and hence, lambs born in September/October are weaned during the dry period, which is characterized by heat stress and a lack of adequate feed quality and quantity, which results in a low growth rate of lambs [Another significant factor that influenced the Ywt was the lambing season. On the 365th day of life, lambs born in April/May were considerably (of lambs . TherefoThe RedM lambs’ higher post-weaning performance may be an indication of early resilience and adaptation to local environmental variables independent of maternal influence. This underlines once more the fact that native breeds of sheep cannot be entirely replaced by foreign breeds, notwithstanding the desire for superior growth performance in exotic breeds. This study’s findings indicate that there may be a genetic benefit from within-breed selection, which is consistent with Kenya’s efforts to maintain the native RedM breed. Dorper sheep should only be used as terminal sires to take advantage of heterosis. The breeding program should be planned to maximize the growing performance of the lambs while considering any effects of potential fixed factors on the desired trait. The establishment of a better and more precise conservation effort should also include genetic diversity and genomic research on the probable genomic variations underlying phenotypic performance, including adaptation. The nutritional profile of the available natural pasture should be defined at the paddock level to ensure proper pasture usage.


The influence of maternal levothyroxine treatment during pregnancy remains unclear. This study aimed to evaluate the associations of maternal levothyroxine treatment during pregnancy with the birth and neurodevelopmental outcomes in offspring.This population-based cohort study was conducted among pregnant women using the Hong Kong Clinical Data Analysis and Reporting System. Mother-child pairs in Hong Kong from 2001 to 2015 were included and children were followed up till 2020. We defined the exposure group as mothers who were exposed to levothyroxine during pregnancy. Preterm birth and small for gestational age (SGA) were included as birth outcomes. Attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) were included as neurodevelopmental outcomes. Odds ratios (OR) or hazard ratios (HRs) with a 95% confidence interval (CI) were evaluated to assess the association of gestational levothyroxine use with offspring birth and neurodevelopmental outcomes respectively, using propensity score fine-stratification weighting and a Cox proportional hazards regression model.Among 422,156 mother-child pairs, 2125 children were born from mothers exposed to levothyroxine during pregnancy. A significantly increased risk of preterm birth was observed in children with maternal levothyroxine exposure during pregnancy, when compared to mothers who had no history of thyroid-related diagnoses or prescriptions . Similarly, an increased risk of preterm birth was found among children of gestational levothyroxine users, when compared to children of mothers who had used levothyroxine before but stopped during pregnancy . Sensitivity analysis, by excluding mothers exposed to psychotropic or antiepileptic medications before or during pregnancy, also indicated a similar increased risk of preterm birth regarding the gestational use of levothyroxine . No significant association was observed for the risk of SGA, ADHD, and ASD.There is no evidence that gestational use of levothyroxine is associated with SGA, ADHD, or ASD in offspring. Gestational levothyroxine treatment is associated with a higher risk of preterm birth. Such risk might be confounded by the underlying maternal thyroid disease itself, however, we cannot completely exclude the possible effect of gestational L-T4 treatment on offspring preterm birth. Our findings provided support to the current guidelines on the cautious use of levothyroxine treatment during pregnancy.The online version contains supplementary material available at 10.1186/s12916-022-02586-9. Thyroid diseases are the second most common endocrine disorder in pregnancy, which are broadly categorized into hyperthyroidism and hypothyroidism, with a prevalence of around 0.2% and 2.5%, respectively –6. As thThough previous studies have suggested the association of maternal hypothyroidism with increased risk of neurodevelopmental disorders of the offspring , little Considering the inconsistency and uncertainty of the findings in previous studies, this study aimed to estimate the associations of maternal L-T4 treatment during pregnancy with birth and neurodevelopmental outcomes in offspring using a large real-world cohort of pregnant women in Hong Kong.This was a territory-wide retrospective cohort study using data from the Hong Kong Clinical Data Analysis and Reporting System (CDARS), which is operated by the Hospital Authority (HA) that manages all public hospitals and the associated ambulatory clinics in Hong Kong. CDARS contains electronic medical records of over 7.5 million Hong Kong residents, including their demographics, diagnosis, and prescription records. The database has been used for various high-quality epidemiological studies –25, inclThis study included mother-child pairs in Hong Kong from 1st January 2001 to 31st December 2015. The children were followed up till 31st December 2020, resulting in at least 5 years of follow-up time. We defined a valid mother-child linkage as an exact match of mother and child identification numbers, delivery date, and delivery hospital. Given that the mother-child linkage is created deterministically by the HA for clinical management where the mother and the child records are linked permanently immediately after delivery, this linkage is considered highly accurate . All theWe defined L-T4 users as mothers with ≥2 dispensing records for thyroid hormone treatment  before giving birth. In Hong Kong, anti-thyroid drugs are the first-line therapy for hyperthyroidism treatment, while thyroidectomy or radioactive iodine (RAI) therapy are the alternatives when anti-thyroid drug treatments fail. Patients receiving thyroidectomy/RAI often require the prescription of L-T4 afterwards when overt hypothyroidism develops. Therefore, we excluded mothers exposed to anti-thyroid drugs, thyroidectomy, or RAI during the study period to make sure that mothers in the exposure group have relatively similar thyroid status and to avoid potential misclassification.gestational users) to those with euthyroid mothers who have never been exposed to L-T4 and had no history of thyroid-related disorders (euthyroid control). Thyroid-related disorders are defined by the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) codes 242-244 in the mother’s longitudinal medical records. The look-back period started from: (1) the beginning of the electronic health record set up in 1995 or (2) the birth of the mother or (3) the first time the mother used HA services. For gestational users, we calculated the median of gestational weeks that mothers started their L-T4 treatments in order to compare with previously published studies.We defined the pregnancy period as the period between the last menstrual period (LMP) and the date of delivery , and neurodevelopmental outcomes including ADHD and ASD. Preterm birth was defined as the delivery of babies before 37 gestational weeks. SGA was defined as offspring whose birth weight was <2 standard deviations (SD) below the mean of the same gestational age. ADHD was defined using an ICD-9-CM record of 314 or a prescription for methylphenidate or atomoxetine. ASD was defined using an ICD-9-CM record of 299. CDARS contains hospital diagnoses from specialists, so the positive predictive value of the neurodevelopmental outcomes is high. The database has previously been used to study ADHD and ASD in children and has been accepted to be a valid database –30.For neurodevelopmental outcomes, the follow-up in children started on the date of delivery and ended on the date of occurrence of the outcome, date of death, or 31 December 2020, whichever came first.Data on maternal comorbidities were obtained from medical records in CDARS. Covariates for adjustment were based on previous literature –34, inclWe performed Propensity Score (PS) fine-stratification weighting to address the differences in baseline covariates. PS methods have advantages over conventional covariate adjustment and have been increasingly applied to adjust for confounding in observational studies . PS fineWe presented proportions for all the outcomes among different comparison groups and PS-weighted cumulative incidence of the neurodevelopmental diagnoses. We presented PS-weighted odds ratios (wORs) with 95% confidence interval (CI) using logistic regression models to assess the association of maternal L-T4 use with birth outcomes of the offspring, and PS-weighted hazard ratios (wHRs) with 95% CI using Cox proportional hazard regression models to assess the association of maternal L-T4 use with neurodevelopment outcomes in offspring. Robust standard errors were used to adjust for the clustering of individuals bound by the same biological mother.pre-pregnancy users) and calculated the median time from pre-pregnancy users’ last prescription of L-T4 till their LMP. We adapted the analytical strategy from a previous study on maternal drug exposure and neurodevelopmental disorders to develop our sensitivity analysis by comparing gestational users to pre-pregnancy users [gestational users compared to euthyroid control, it could be due to the use of levothyroxine and/or presence of hypothyroidism. If there is no difference or a decreased risk in gestational users when compared to pre-pregnancy users, then it suggests that the association may be due to confounding by maternal hypothyroidism. Second, since maternal exposure to psychotropics such as antidepressants, antipsychotics, lithium, and antiepileptic medications may increase the risk of giving birth to a child with birth and neurodevelopmental problems [Additional analyses were conducted to test the validity and robustness of the initial analyses. First, we identified children with mothers exposed to L-T4 before pregnancy but stopped the treatment when pregnant  v9.4  and R . The results were cross-checked for accuracy and consistency by GG and EC.The original data included 422,851 mother-child pairs from 2001 to 2015. Five hundred two perinatal death cases and 193 mother-child pairs with missing gestational age and birth weight were excluded. Then, after removing mothers exposed to anti-thyroid drugs, underwent thyroidectomy or RAI therapy during the study period, and mothers first exposed to L-T4 after giving birth, the cohort included 401,207 mother-child pairs. There were 2125 children born from mothers who received L-T4 during pregnancy, and 398,909 children born from mothers who had no history of thyroid-related diagnoses or prescriptions. A total of 173 children were born from mothers exposed to L-T4 before pregnancy but stopped the treatment when pregnant  and 10.60 (SD=4.20) years in children of gestational L-T4 users, and 10.75 (SD=3.87) and 10.77 (SD=3.95) years in children of euthyroid control mothers , but not for SGA . In terms of neurodevelopmental outcomes, no significant association was observed for the risk of ADHD and ASD between children of gestational L-T4 users and euthyroid control mothers, with a wHR of 1.10  and 1.00 , respectively , but no statistically significant difference was observed for SGA, ADHD, and ASD , and a null association was observed for SGA, ADHD, and ASD.Sensitivity analysis by restricting the analyses to a subgroup of children born from mothers without exposure to psychotropics or anti-epileptics before or during pregnancy showed a consistent pattern of results . For SGA, ADHD, and ASD, the estimates were higher in boys compared to that in girls, but no statistically significant difference was observed between children of gestational L-T4 users and euthyroid control.Subgroup analysis by stratifying offspring sex suggested a similar pattern of results . The results remained unchanged after adjusting maternal pre-eclampsia in the PS fine stratification model. . The increased risk of preterm birth was also observed in the after 2011 group, but the estimate was marginally significant . For SGA, ADHD, or ASD, no significant difference was observed between children of gestational L-T4 users and euthyroid control. The interaction analysis suggests that the periods before and after the year 2011 did not affect the estimates in the comparison between gestational L-T4 users and euthyroid control  mothers with normal to elevated levels of thyroid-stimulating hormone found no difference in preterm birth and miscarriage rate between mothers with and without L-T4 treatment during pregnancy . HoweverTo our knowledge, no previous study focused on investigating the association of maternal L-T4 use during pregnancy with the risk of both birth and neurodevelopmental outcomes in children. Only one placebo-controlled randomized trial was specifically designed to examine the efficacy of L-T4 treatment in pregnant women with subclinical hypothyroidism/hypothyroxinemia . PsycholOur sensitivity analysis suggested that offspring sex does not influence the overall conclusion from the result. Though no significant association was observed between children with gestational L-T4 mothers and euthyroid mothers, the estimates of SGA and neurodevelopmental disorders were higher in boys than in girls. This finding was consistent with previous studies regarding ADHD and ASD among girls and boys , 48.The current study had several strengths. First, the study analyzed a large population-based sample available as electronic health records from CDARS, with highly accurate and reliable information on the mother-child link. In addition, automated dispensing and prescribing records were applied to identify exposures and neurodevelopmental outcomes, which are free of recall bias. Second, our study involved longitudinal data and investigated both birth and neurodevelopmental outcomes associated with significant morbidity and mortality. Third, several sensitivity analyses suggested that the results from the main analysis were robust and reliable.However, there were limitations. First, CDARS only contains medical records from publicly funded hospitals but not private hospitals and medical practitioners. In this study, we captured all birth events that occurred in public hospitals, which account for about 66% of births in Hong Kong. Though the remaining 34% in private healthcare were not covered in the current study, it is unlikely to affect the interpretation of our results as all comparisons were conducted in patients using HA services. In addition, the public sector is the main provider of medical care for neurodevelopmental disorders in Hong Kong. As children diagnosed with ADHD or ASD usually require long-term monitoring and treatment, they usually utilize health services provided by the public sector. Thus, they are most likely to be captured in this study. Second, exposure to L-T4 is defined by prescribing and dispensing records. Poor medication adherence could have biased the results towards the null. Nevertheless, we identified those with at least two L-T4 prescribing records to ameliorate this potential bias. Third, in Hong Kong, subclinical hypothyroidism (SCH) is not recommended to be treated. In this study, mothers who received L-T4 treatment were clinically diagnosed with hypothyroidism and were involved in routine clinical care. The interpretation of this study may not apply to individuals with SCH. Nevertheless, this study aimed to estimate the effect of gestational L-T4 treatment on offspring. Fourth, we could not tease apart the effects of hypothyroidism (including under-treated hypothyroidism) or gestational L-T4 treatment on offspring preterm birth as we did not account for thyroid hormone levels in the analysis. We recommend future research to adopt thyroid hormone lab test information and address the issues regarding disease severity and all forms of thyroid dysfunction. Fifth, though we applied different cut-offs considering the severity of preterm birth, we were not able to differentiate between spontaneous or medically indicated preterm birth. Sixth, given the nature of observational designs, all sources of confounding cannot be fully ruled out. We did not adjust the socioeconomic status of mothers in the cohort such as smoking or alcohol. To address potentially measured and unmeasured confounding, this study adopted additional sensitivity and subgroup analysis to help rule out some but not all sources of confounding to provide complementary evidence.There have been frequent updates in the guidelines of the American Thyroid Association (ATA) for L-T4 use in pregnant women in recent years. In 2011, ATA accepted thyroid-stimulating hormone of 2.5 mIU/L as the upper limit of the normal level in the first trimester . CliniciThe present study suggested that gestational exposure to L-T4 was associated with an increased risk of preterm birth but not SGA, ADHD, or ASD. Though we cannot completely exclude the possible effect of gestational L-T4 treatment on offspring preterm birth, such risk might be confounded by the underlying maternal thyroid disease itself. Decision-making about L-T4 use in pregnancy remains important and requires an assessment of the risks and benefits in the context of both mothers and children.Additional file 1: Figure S1. Pregnancy period and comparison groups’ identification. Figure S2. Cumulative incidence of ADHD and ASD by maternal L-T4 exposure. Figure S3. Box plot of the days between last L-T4 prescription and LMP of pre-pregnancy users. Figure S4. Box plot of cumulative dose among the gestational users during pregnancy. Figure S5. Box plot of length of time the mothers use L-T4 before pregnancy.Additional file 2: Table S1. ICD-9-CM codes applied for covariates. Table S2. Covariates balance for comparison between gestational L-T4 users and euthyroid control. Table S3. Percentage of gestational users who started L-T4 treatment at different trimesters. Table S4. Covariates balance for the comparison between gestational users and pre-pregnancy users. Table S5. Sensitivity analysis by comparing gestational L-T4 users to pre-pregnancy. Table S6. Sensitivity analysis by excluding mothers exposed to psychotropic or antiepileptic medications before or during pregnancy. Table S7. Subgroup analysis by stratifying offspring sex. Table S8. Sensitivity analysis by using different SGA definitions. Table S9. Sensitivity analysis by different cut-offs for preterm birth. Table S10. Sensitivity analysis for the risk of offspring ADHD by restricting to children born before the year 2014. Table S11. Comparison between gestational L-T4 users before and after year 2011. Table S12. Interaction analysis of before and after year 2011 on the estimates in the comparison between gestational L-T4 users and euthyroid control. Table S13. Post-hoc analysis adjusted cumulative dose of L-T4 during pregnancy. Table S14. Post-hoc analysis adjusted length of time the mothers using L-T4 before pregnancy.


Background: Salivary cortisol (SC), a commonly used biomarker for stress, may be disrupted by negative events in pregnancy, at birth and in infancy. We aimed to explore if maternal perceived stress (PSS) in or after pregnancy and SC levels in pregnancy were associated with SC in early infancy, and, secondly, to identify early life factors associated with infants’ SC levels (iSC). Methods: At 3 months of age, SC was analyzed in 1057 infants participating in a Nordic prospective mother-child birth cohort study. Maternal PSS was available from questionnaires at 18- and 34-week gestational age (GA) and 3-month post-partum, and SC was analyzed at 18-week GA. Early life factors included sociodemographic and infant feeding from questionnaires, and birth data from medical charts. Associations to iSC were analyzed by Spearman correlation and multinomial logistic regression analyses. Results: In this exploratory study neither PSS at any time point nor maternal SC (mSC) were associated with iSC. Higher birth weight was associated with higher levels of iSC, while inverse associations were observed in infants to a mother not living with a partner and mixed bottle/breastfeeding. Conclusions: Maternal stress was not associated with iSC levels, while birth weight, single motherhood and infant feeding may influence iSC levels. Salivary cortisol (SC) is a commonly used biomarker for stress in infants and children ,2,3. An In shared activities during the first year between mother and infant , SC levels can fluctuate in the infant and covary between the two ,8. NegatThe aims of this exploratory study were to analyze the associations between maternal perceived stress during pregnancy and postpartum as well as pregnancy mSC levels and iSC levels at three months of age, and secondly to identify early life factors in pregnancy, at birth and in infancy that are associated with iSC levels at three months of age.The women and infants in the present study are participants in the multicenter interventional birth cohort study PreventADALL (Preventing Atopic Dermatitis and ALLergy in children), conducted at the Oslo University Hospital and the Østfold Hospital Trust, Oslo, Norway; and Karolinska University Hospital, Stockholm, Sweden . The PreAt enrolment, a short interview was performed, height, weight and blood pressure were measured, a blood sample was drawn and a home saliva sampling kit was provided to the pregnant women. Electronic questionnaires were distributed twice in pregnancy; first at enrolment and the second around pregnancy week 34, based largely on questionnaires used in previous birth cohort studies ,20,21. TFrom the 2697 pregnant women and their 2397 newborns enrolled in the PreventADALL study, three withdrew their consent, leaving 2394 mother-child pairs, whereof 2131 infants attended the three-month clinical examination. In the present study we included all 1057 infants who had available iSC measurement from saliva samples collected at three months of age .®, Carlsbad, CA, USA), a form to fill out the sampling time and date and prepaid addressed envelope to the study center (in Oslo for the Norwegian participants and in Stockholm for the Swedish participants). Parents were instructed to collect saliva within the next days, preferably on Monday, Tuesday or Wednesday, shortly after their own or the infant’s awakening (after 06.00) and before food/breastfeeding or water intake. The sample was refrigerated and mailed to the study center as soon as possible. Cortisol concentrations have previously shown stability in the postal service and to exposure to different temperatures [Home sampling kits were distributed at inclusion in pregnancy for maternal saliva sampling and at the three-month follow-up visit for infant saliva sampling. All kits included sampling instructions, a zip-locked plastic bag, a saliva kit , according to the manufacturer’s instructions, with CORT-CT2 kits from Cisbio Bioassays  at Forskningscentrum  . SalivarInfant SC samples with a stated sampling time were collected between 02 AM and 9 PM . The maiInformation on maternal stress was included in the electronic questionnaires and was measured with Cohen’s Perceived Stress Scale (PSS) (14 items), which scores from 0 to 4 for each of the 14 items with a total maximum score of 56 , and meaEarly life factors collected through the electronic questionnaire in mid-pregnancy, included socio-demographic information, previous and current health and maternal stress, while information about the father and socio-demographic, health or disease changes in the mother since inclusion was captured in the 34-week GA questionnaire. The information included mothers not living with a partner; e.g., women who lived alone, divorced, widow, other, low maternal education (maximum 9/10-year school or high school), maternal age at inclusion and if it was the first pregnancy or the mother had previous deliveries .Birth-related factors were collected from the maternal and infant birth records and by PreventADALL study personnel at the inclusion visit of the infant. Induced labor was defined as a medically induced labor. Vaginal birth included unassisted and births assisted birth with forceps or vacuum extraction. Cesarean section included planned elective and acute cesarean section. Birth weight was the infant’s first recorded weight.Infant factors included exclusively breastfed and mixed feeding by bottle and breastfeeding (mixed fed) as well as colic, abdominal pain and pain or other discomforts reported at three months of age based on the following question: “In the last three months, did the infant have any of following?” . Furtherp-value.Infant SC levels included some outliers (data were positively skewed). Thus, descriptive statistics are presented with median, interquartile range (IQR) or minimum-maximum (min-max) (continuous variables) and counts and percentages (%) . Spearman non-parametric correlations were used to describe correlations between infant SC levels and maternal PSS and SC levels and are presented with Spearman’s coefficient rho (r) and n = 266), 5.51–15.35 nmol/L (n = 263), 15.36–24.93 nmol/L (n = 265) and ≥24.94 nmol/L (n = 263). In the univariate and multivariate multinomial logistic regression models, the group with the lowest iSC levels (≤5.50 nmol/L) was used as the reference category. Variables with p-value less than 0.1 from univariate analyses , multinomial logistic regression was used. Infant SC data were divided in four equally large groups ≤5.50 nmol/L , 13.20 nmol/L (IQR 16.1) in the 79 with other sampling time and 17.70 nmol/L (IQR 18.30) in the 427 infants with missing sampling time  at three months of age and had a median age of 14 weeks and 51.4% were boys . The oveing time .Maternal PSS was not significantly associated with iSC, neither by PSS in mid-trimester, third trimester or postnatally, nor among all infants (main outcome) or by sampling time .Maternal SC levels in mid-pregnancy was not significantly associated with iSC levels among all infants, nor among infants with morning sampling . Furtherp-values were ≤0.1, while not living with a partner, birth weight and mixed-fed infants remained statistically significantly associated with iSC in the final model  and missing records of sampling time of the infant saliva due to the home-sampling procedure. All iSC were analysed without any consideration of sampling time and skewness. SC samples were not tested for blood contamination. Our participants were instructed to collect the saliva sampling in the morning, 30 min after the child’s or own awakening, preferably before breast- or bottle-feeding. We did not collect complementary information nor further investigate if the instructions to study protocol were followed. Our stress data were based on one validated instrument and it was collected through self-reported electronic questionnaires which could increase the risk of information bias. PSS has been translated but not validated into the Norwegian language. Several samples in our study were taken later in the morning, in the afternoon or had a missing sampling time. We did not see, nor could draw conclusions of, a cortisol circadian rhythm in our infant population, neither on a group level nor on an individual level as our study only generated one sample per infant. The intention was to include a general population in this study; however, socioeconomic status was higher among the infants’ families who provided a saliva sample than those who did not (not presented), and data on maternal depression- and anxiety-medication were not available for the mothers. The association between iSC in non-cohabitant mothers was based on a very limited number of individuals, thus might be sporous. Limited statistical power did not allow us to estimate these associations with sufficient precision. All analyses in this study were considered exploratory so no correction for multiple testing was performed. These findings are therefore explorative and need to be confirmed in other studies with similar sampling and settings.Our results suggest that the possibility of identifying a correlation between PSS and iSC could be dependent on the timespan between maternal/infant samples as well as the number of samples taken at each timepoint.In this exploratory study, maternal perceived stress in pregnancy and post-partum was not associated with iSC levels at three months of age. Early life factors which were significantly associated with iSC included increased birthweight with increased iSC levels at three months, while infants of mothers not living with a partner and mixed-fed infants had lower levels of iSC. Altogether, these results suggest that the relationship between PSS and mSC levels in mothers and early life factors of iSC levels in infants are complex.