Circulating inflammatory biomarkers and academic performance in adolescents: DADOS study.

OBJECTIVE: The present study aimed (1) to examine the association between circulating inflammatory biomarkers and academic performance in adolescents, and (2) to identify the ability of circulating inflammatory biomarkers to predict low academic performance.
METHODS: A total of 244 adolescents (13.9±0.3 years, 112 girls) from the DADOS study were included in the analysis. Four inflammatory biomarkers were quantified: white blood cell (WBC) count, interleukin-6, tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP). Academic performance was assessed through academic grades and the Spanish version of the Science Research Associates Test of Educational Abilities.
RESULTS: TNF-α was inversely associated with math, Spanish and grade point average (β ranging from -0.166 to -0.124; all p<0.05), while CRP was inversely associated with verbal ability (β = -0.128; p<0.05). Overall, receiver operating characteristic (ROC) curves analyses showed discriminatory ability of WBC and TNF-α in identifying low academic performance (all p<0.05). Moreover, logistic regression analyses indicated that students with levels of WBC and TNF-α above the ROC cut-offs values showed between 78% to 87% increased likelihood of lower academic performance (p<0.05).
CONCLUSIONS: Our findings suggested that some circulating inflammatory biomarkers were associated with academic performance in adolescents. Further larger longitudinal and interventional studies are needed to clarify the short-term and long-term relationship between inflammation and academic performance in youths.

Introduction

Inflammation is a natural immune system response to injury, infectious agent, or oxidative stress. This mechanism can confer immune protection, promoting tissue survival, repair, and recovery. However, a prolonged activation of the peripheral immune system could lead to a state of systemic low-grade inflammation [1]. Prior research has suggested systemic low-grade inflammation to be both a cause, and a consequence of pathological processes related to several cardiovascular and metabolic diseases (e.g., atherosclerosis, diabetes, cancer) [2, 3], as well as to the development of neuropsychiatric disorders [4].

Emerging evidence indicates that circulating inflammatory biomarkers might also play a key role on cognition during the early and late stages of human lifespan [5, 6]. In fact, most of the studies reporting an inverse association between circulating inflammatory biomarkers and cognition have been focused on preterm infants [7, 8], and aging populations [9, 10], as well as in populations with neuropsychiatric disorders [11]. However, few studies have investigated the link between inflammation and cognitive function in adolescents, showing controversial results since not only negative [12], but also null [13] associations have been found.

Cognition may be closely linked to academic performance, which has shown to predict future health status [14] and work opportunities [15]. However, only one study has analysed the association between inflammation and academic performance in adolescents, showing an inverse association between circulating inflammatory biomarkers and academic grades [16].

Given the influence that inflammation may have on cognition, and the importance of academic performance for adolescents’ future, it is of particular interest to clarify if inflammatory biomarkers are associated with academic performance in this age group. Thus, the present study aimed (1) to examine the association between circulating inflammatory biomarkers and academic performance in adolescents, and (2) to identify the ability of circulating inflammatory biomarkers to predict low academic performance.

Materials and methods

Study design and sample selection

The present work is part of the DADOS (Deporte, ADOlescencia y Salud) study, a research project aimed to analyse the influence of health-related factors on health and academic performance in adolescents [17]. The results presented in this study belong to baseline data obtained between March and May of 2015. A convenience sampling technique was used to recruit participants. For that purpose, advertising leaflets about the research project were sent to secondary schools and sport clubs located in Castellon (Spain). These included basic information and the general inclusion criteria of DADOS study which were: to be enrolled in 2nd grade of secondary school (i.e., the 8th grade), not having failed a previous academic year, and without having any medical diagnosis of physical or mental illness (as reported by participants’ parents or guardians). Volunteers who met the inclusion criteria contacted the research group and were included in the study. We estimated that a sample of 300 participants would be required to provide statistical power of 80% with a level of significance of 0.05, assuming a dropout rate of 20%. Finally, from the total DADOS study sample (n = 274), 244 adolescents (112 girls) were included in the analyses. This final sample came from 38 secondary schools, out of 85 located in the province of Castellon (from which 19 were private schools, out of 34 located in this province), and had valid data for at least one circulating inflammatory biomarker and academic performance. All participants included in the current analyses self-reported before blood sample collection that they were not suffering from acute illnesses (e.g., flue, infection, fever, allergies, toothache) at the time of testing either during the last week.

Students and their parents or guardians were informed of the nature and characteristics of the study, and all provided written informed consent. The study protocol was designed in accordance with the ethical guidelines of the 1961 Declaration of Helsinki (last revision of Fortaleza, Brazil, 2013) and approved by the Research Ethics Committee of the Jaume I University of Castellon.

Circulating inflammatory biomarkers

Blood samples were drawn from the antecubital vein after an overnight fast of at least 10 h (at 8:00 a.m.), and collected in two tubes containing EDTA (Greiner bioone, Kremsmünster, Austria). One tube was kept refrigerated at 4°C for immediate analyses in whole blood, while the other tube was centrifuged to obtain serum (3500 rpm for 10 min at 4°C). The following inflammatory biomarkers were quantified: white blood cell (WBC, 103/μL) count, interleukin-6 (IL-6, pg/mL), tumor necrosis factor-α (TNF-α, pg/mL), and C-reactive protein (CRP, mg/dL). WBC count was measured in whole blood by automated blood cell counters (ABX Pentra XL 80, Horiba ABX SAS; Montpellier, France) with an intra-assay precision coefficient of variation (CV) of <2%. IL-6 and TNF-α were determined in serum using specific sensitive Enzyme-Linked Immunosorbent Assay (ELISA) kits (DRG Instruments GmbH, Marburg, Germany) with a sensitivity of 2 pg/mL for IL-6, and 0.7 pg/mL for TNF-α. The intra- and inter-assay precision CVs were 4.2% and 4.4% for IL-6, and 6.6% and 4.5% for TNF-α, respectively. The CRP concentration was quantified in serum by immunoturbidimetry (CRP 981699, Thermo Fisher Scientific Oy; Vantaa, Finlandia) with a sensitivity of 6 pg/mL and intra- and inter-assay CVs of 2.6% and 0.8%, respectively.

Academic performance

Academic performance was assessed using academic grades and a standardized test of academic abilities. Academic grades were taken from the participants’ official report cards obtained at the end of the academic year, which were provided by parents or guardians. Individual grades for math, Spanish and English, as well as the grade point average score were included in the analyses. The grade point average score was calculated as the single average for geography and history, natural sciences, maths, Spanish, Catalan and English languages and physical education grades. All the subjects were measured on a ten-point scale, where 0 was the worst and 10 was the best. Academic abilities were assessed through the Spanish version of the Science Research Associates Test of Educational Abilities [18], which was completed within the same week that blood samples were collected. This test measures three basic academic abilities: verbal ability (command of language), numeric ability (speed and precision in performing operations with numbers and quantitative concepts), and reasoning ability (the aptitude to find logical ordination criteria in sets of numbers, figures, or letters). Scores for the three abilities were obtained by adding positive answers. Overall academic ability was calculated by adding the three abilities’ scores (verbal + numeric + reasoning). The present study used level three, which is designed for adolescents aged 14–18 years. The alpha scores for its reliability have been reported to be 0.74 for verbal ability, 0.87 for numerical ability, 0.77 for reasoning ability and 0.89 for overall academic ability [18]. Participants were classified in high academic performance (≥50th of the median) and low academic performance (<50th of the median) for each academic performance indicator.

Covariates

The statistical analyses were controlled for sex, pubertal stage, socioeconomic status related variables (i.e., parental educational level and type of school), waist circumference, and adherence to the Mediterranean diet. These are relevant cofounders given the association of socioeconomic status [19], waist circumference [20, 21] and adherence to the Mediterranean diet [17, 22] with inflammation and academic performance. In addition, since adolescence is a period of developmental changes at a different pace, sex and pubertal stage were also considered as covariates.

Pubertal stage

Pubertal stage was self-reported according to the five stages described by Tanner [23] based on the assessment of two components: pubic hair growth for boys and girls, plus breast development in girls, and genital development in boys. A 5-point maturity rating was used where stage 1 corresponds to the prepubertal state and stage 5 to mature state, and the highest rating of the two components was used for the analyses.

Parental educational level

Parental educational level was used as a proxy of socioeconomic status [24]. Both parents reported their educational level and responses were combined as: neither of the parents had a university degree, and at least one of the parents had a university degree.

Type of school

Students’ school type was classified into ‘public’ or ‘private’ school, and entered as a dummy variable.

Anthropometry

Measures were assessed in duplicate by experienced researchers following standardized procedures [25], and average measures were used for the analyses. Briefly, body weight was measured to the nearest 0.1 kg using an electronic scale (SECA 861, Hamburg, Germany). Height was measured to the nearest 0.1 cm using a wall-mounted stadiometer (SECA 213, Hamburg, Germany). Body mass index (BMI) was calculated as weight/height squared (kg/m2). Participants were classified into normal weight and overweight or obese, according to the international age- and sex-specific BMI cut-offs proposed by Cole et al. [26]. Waist circumference was measured, as a proxy of abdominal obesity, to the nearest 1 mm with a non-elastic tape applied horizontally midway between the lowest rib margin and the iliac crest, at the end of gentle expiration with the adolescent in a standing position.

Adherence to the Mediterranean diet

Adherence to the Mediterranean diet was evaluated using the KIDMED questionnaire, which includes 16 yes/no questions related to participants consumption of fast food, sweets and soft drinks, daily fruit and vegetables, and weekly fish and legumes [27]. Regarding the affirmative answers, a value of +1 was assigned to the questions with positive connotation in relation to Mediterranean diet (e.g., regular fruit consumption), while a value of -1 was assigned to the questions that constitute negative aspects (e.g., fast food consumption). Questions answered with “no” scored 0. The score for the students’ level of adherence to the Mediterranean diet was calculated as the sum of each answer, ranging from 0 to 12.

Statistical analysis

Descriptive characteristics of the study sample are presented as means ± standard deviation or frequency (%). Differences between sexes were examined by independent two-tailed t-tests and Chi-squared tests for continuous and categorical variables, respectively. All variables were checked for normality using both graphical (normal probability plots) and statistical (Kolmogorov-Smirnov test) procedures. Due to its skewed distribution, circulating inflammatory biomarkers were log-transformed when required. As preliminary analyses showed no significant interactions of sex with circulating inflammatory biomarkers in relation to academic performance indicators (all p>0.1), all analyses were performed with the total sample.

Linear regression analyses were used to study the association between circulating inflammatory biomarkers and academic performance indicators adjusting for sex, pubertal stage, parental educational level, type of school, waist circumference and adherence to the Mediterranean diet.

Receiver operating characteristic (ROC) curves were conducted to investigate the ability of circulating inflammatory biomarkers (i.e., WBC, IL-6, TNF-α, and CRP) in discriminating low academic performance. The area under the curve (AUC) ranges between 0 and 1, where 0 represents a worthless test, and 1 a perfect ability of circulating inflammatory biomarkers to identify students with low academic performance. When the AUC was statistically significant, cut-off points were selected according to the highest Youden index, which is calculated with the best trade-off between sensitivity and specificity.

Based on the ROC curves analyses, logistic regression analyses were conducted to examine the relationships between high circulating inflammatory biomarkers concentrations (i.e., ≥cut-off values) and low academic performance, adjusting for sex, pubertal stage, parental educational level, type of school, waist circumference and adherence to the Mediterranean diet. These analyses were performed only for those circulating inflammatory biomarkers that showed a discriminatory ability to predict low academic performance (AUC>0.5 and p<0.05). All the analyses were performed using the IBM SPSS Statistics for Windows version 22.0 (Armonk, NY: IBM Corp), and the level of significance was set to p<0.05.

Results

Sample characteristics

Table 1 summarizes adolescents’ characteristics by sex. Our study included 244 adolescents aged 13.9 ± 0.3 years old, of which 112 (45.9%) were girls. Boys presented higher values in height (164.7 vs. 160.9; p<0.001), waist circumference (68 vs. 66; p<0.01), TNF-α (5.6 vs. 4.8; p<0.01), and numeric ability (14.9 vs. 12.4; p<0.001) than girls.

Table 1

Descriptive characteristics for the study sample.

	All	Boys	Girls	p
n (%)	244 (100)	132 (54)	112 (46)
Age (y)	13.9 ± 0.3	13.9 ± 0.3	13.9 ± 0.3	0.709
Pubertal stage (I-V) (%)	8/35/47/10	9/33/43/15	0/6/38/52/4	-
Height (cm)	163.0 ± 8.1	164.7 ± 8.6	160.9 ± 7.0	<0.001
Weight (kg)	53.8 ± 9.1	54.4 ± 9.3	53.1 ± 8.8	0.297
Body mass index (kg/m2)	20.2 ± 2.6	19.9 ± 2.4	20.5 ± 2.8	0.106
Overweight and obesity, n (%)	29 (12)	15 (11)	14 (13)	0.785
Waist circumference (cm)	67.1 ± 5.6	68.0 ± 5.2	66.0 ± 5.8	0.006
Adherence to the Mediterranean diet (0–12)	7.1 ± 2.1	7.3 ± 2.2	6.8 ± 2.1	0.059
Parental educational level
University studies, n (%)	118 (48)	58 (44)	60 (54)	0.134
School type
Private (%)	67 (28)	41 (31)	26 (23)	0.171
Circulating inflammatory biomarkers a
White blood cells (103/μL)	5.7 ± 1.4	5.6 ± 1.3	5.8 ± 1.4	0.426
Interleukin-6 (pg/mL) (n = 206)	3.1 ± 3.0	3.1 ± 3.1	3.1 ± 2.9	0.975
Tumor necrosis factor-α (pg/mL)	5.2 ± 2.2	5.6 ± 2.3	4.8 ± 2.1	0.004
C-reactive protein (mg/dL)	0.53 ± 0.21	0.54 ± 0.25	0.52 ± 0.14	0.312
Academic grades (0–10)
Math	6.53 ± 1.7	6.5 ± 1.7	6.7 ± 1.7	0.423
Spanish	6.7 ± 1.7	6.5 ± 1.7	7.0 ± 1.7	0.014
English	6.9 ± 1.7	6.7 ± 1.7	7.2 ± 1.7	0.025
Grade point average	6.9 ± 1.4	6.8 ± 1.3	7.0 ± 1.4	0.136
Academic abilities
Verbal (0–50)	19.0 ± 5.4	19.2 ± 6.0	18.8 ± 4.5	0.579
Numeric (0–30)	13.8 ± 4.7	14.9 ± 4.6	12.4 ± 4.4	<0.001
Reasoning (0–30)	16.7 ± 5.8	16.2 ± 5.7	17.3 ± 5.8	0.131
Overall (0–110)	49.5 ± 12.4	50.3 ± 13.0	48.5 ± 11.7	0.262

Data are presented as mean ± standard deviation or frequency (%). Differences between sexes were examined by t test or chi-square test. Statistically significant values are in bold.

a Values were log-transformed before analysis, but non-transformed values are presented.

Association between inflammatory biomarkers and academic performance

Linear regression analyses between circulating inflammatory biomarkers and academic performance indicators after adjustment for sex, pubertal stage, parental educational level, type of school, waist circumference, and adherence to the Mediterranean diet are shown in Tables 2 and 3. WBC and IL-6 were not associated with academic performance indicators. TNF-α was negatively associated with math (β = -0.166; p<0.01), Spanish (β = -0.127; p<0.05), and grade point average (β = -0.124; p<0.05), while CRP was negatively associated with verbal ability (β = -0.128; p<0.05).

Table 2

Linear regression analysis examining the association between circulating inflammatory biomarkers and academic grades.

	Math			Spanish			English			Grade point average
	β	95% CI	p	β	95% CI	p	β	95% CI	p	β	95% CI	p
White blood cells	-0.072	-3.395; 0.894	0.252	-0.058	-3.152; 1.140	0.357	-0.108	-3.888; 0.246	0.084	-0.066	-2.544; 0.742	0.281
Interleukin-6	0.020	-0.816; 1.110	0.765	-0.042	-1.297; 0.670	0.532	0.053	-0.535; 1.263	0.426	0.009	-0.692; 0.790	0.897
Tumor necrosis factor-α	-0.166	-2.201; -0.337	0.008	-0.127	-1.906; -0.032	0.043	-0.112	-1.752; 0.076	0.072	-0.124	-1.463; -0.027	0.042
C-reactive protein	-0.068	-4.192; 1.199	0.275	-0.082	-4.487; 0.894	0.190	-0.087	-4.470; 0.743	0.160	-0.071	-3.291; 0.835	0.242

β: standardized coefficient. CI: confidence interval. Analyses were adjusted for sex, pubertal stage, parental educational level, type of school, waist circumference, and adherence to the Mediterranean diet.

Table 3

Linear regression analysis examining the association between circulating inflammatory biomarkers and academic abilities.

	Verbal			Numeric			Reasoning			Overall
	β	95% CI	p	β	95% CI	p	β	95% CI	p	β	95% CI	p
White blood cells	-0.041	-8.716; 4.356	0.512	-0.017	-6.332; 4.712	0.773	-0.007	-7.651; 6.820	0.910	-0.027	-18.803; 11.993	0.664
Interleukin-6	0.083	-1.301; 5.104	0.243	0.105	-0.508; 4.512	0.117	0.057	-2.070; 4.834	0.431	0.100	-2.082; 12.652	0.159
Tumor necrosis factor-α	0.020	-2.481; 3.462	0.745	-0.067	-3.978; 1.042	0.250	-0.096	-5.856; 0.717	0.125	-0.061	-10.541; 3.446	0.319
C-reactive protein	-0.128	-17.224; -0.580	0.036	0.015	-6.142; 8.068	0.790	-0.058	-13.685; 4.930	0.355	-0.075	-32.048; 7.415	0.220

β: standardized coefficient. CI: confidence interval. Analyses were adjusted for sex, pubertal stage, parental educational level, type of school, waist circumference, and adherence to the Mediterranean diet.

Diagnostic ability of inflammatory biomarkers to predict low academic performance

Table 4 presents the parameters of the ROC curve analyses regarding the diagnostic ability of circulating inflammatory biomarkers to predict low academic performance in adolescents. ROC analyses showed that IL-6 and CRP concentrations did not discriminate among academic performance categories. However, significant AUC were found for WBC with English, and for TNF-α with all the academic grades indicators (all AUC>0.5 and p>0.05). No circulating inflammatory biomarker showed discriminatory ability to identify low academic abilities (S1 Table).

Table 4

Parameters of the receiver operating characteristic curve analysis for the diagnostic performance of circulating inflammatory biomarkers in identifying low academic grades.

Low academic grades		White blood cells	Interleukin-6	Tumor necrosis factor-α	C-reactive protein
Math	AUC	0.499	0.496	0.582	0.536
	95%CI	0.426–0.572	0.417–0.575	0.510–0.653	0.463–0.609
	p	0.982	0.929	0.028	0.329
	Cut-off	-	-	≥5.75	-
	Sensitivity (%)	-	-	0.713	-
	Specificity (%)	-	-	0.425	-
Spanish	AUC	0.549	0.491	0.604	0.505
	95%CI	0.477–0.622	0.412–0.570	0.533–0.675	0.431–0.579
	p	0.186	0.822	0.005	0.893
	Cut-off	-	-	≥5.85	-
	Sensitivity (%)	-	-	0.732	-
	Specificity (%)	-	-	0.443	-
English	AUC	0.581	0.500	0.585	0.552
	95%CI	0.508–0.653	0.420–0.580	0.513–0.658	0.479–0.626
	p	0.032	0.992	0.023	0.166
	Cut-off	≥5.25	-	≥5.85	-
	Sensitivity (%)	0.493	-	0.721	-
	Specificity (%)	0.654	-	0.442	-
Grade point average	AUC	0.562	0.514	0.598	0.506
	95%CI	0.490–0.634	0.435–0.593	0.524–0.667	0.433–0.579
	p	0.094	0.727	0.010	0.872
	Cut-off	-	-	≥5.85	-
	Sensitivity (%)	-	-	0.727	-
	Specificity (%)	-	-	0.430	-

AUC: area under the curve; CI: confidence interval; Values in bold font indicate statistically significant AUC.

Associations between high levels of inflammatory biomarkers and low academic performance

Fig 1 shows logistic regression analyses for the associations between high circulating inflammatory biomarkers (i.e., above the cut-off values provided by the ROC curve analysis) and low academic performance (i.e., <50th of the median) after adjustment for sex, pubertal stage, parental educational level, type of school, waist circumference, and adherence to the Mediterranean diet. High levels of WBC were associated with low academic performance in English (OR = 1.78; 95%CI:1.00–3.15). In addition, students with high concentration of TNF-α had 79%, 86% and 87% increased odds of achieving low academic performance in math, Spanish, and English, respectively.

Fig 1

Logistic regression model predicting low academic grades according to high concentration of circulating inflammatory biomarkers (above the cut-off values provided by the ROC curve analysis).

Analysis adjusted for sex, pubertal stage, parental educational level, type of school, waist circumference, and adherence to the Mediterranean diet. Values in bold font indicate statistically significant results. OR: Odds ratio; CI: confidence interval. Reference (OR = 1.00): students with low concentration of circulating inflammatory biomarkers. n indicates number of adolescents from the total sample (n = 244) over the cut-off values.

Discussion

The main finding of the present study revealed an inverse association between TNF-α and math, Spanish, and grade point average after adjusting for potential confounders. In addition, CRP was inversely associated with verbal ability. Our study indicates that WBC presented discriminatory ability in identifying low academic performance in English, while TNF-α showed discriminatory ability in identifying low academic performance in all the academic grades analysed. Overall, students with high levels of WBC and TNF-α showed between 78% to 87% increased likelihood of low academic performance. These results further contribute to the scarce prior literature suggesting that inflammation may negatively influence academic performance in healthy adolescents [16].

To the best of our knowledge, no previous studies have examined the association between circulating inflammatory biomarkers and academic abilities, neither their ability for identifying low academic performance, which hampers comparisons among studies. To date, there is only one study that has investigated the association between inflammatory biomarkers and academic grades in adolescents [16]. In contrast to our results, Esteban-Cornejo et al. [16] found that WBC, IL-6 and CRP, but not TNF-α, were inversely associated with academic grades in math, Spanish, the mean of math and Spanish, and grade point average, independently of adiposity, in a sample of Spanish children and adolescents aged 6–18 years.

The reasons underlying the inverse association between TNF-α and academic grades as well as between CRP and verbal ability cannot be elucidated in the current study. However, these findings may be partially related to the key role that these inflammatory biomarkers play on brain functioning. Based on in vitro studies, we speculate that TNF-α could affect academic outcomes by reducing neurogenesis, increasing apoptosis of neurons, and consequently, inhibiting synaptic plasticity and memory consolidation [4]. Interestingly, although much work has been carried out in mice, prior research in humans has suggested that TNF-α-driven processes may contribute to cognitive impairments [6], which could also negatively influence academic results. In addition, a previous study indicated that CRP may negatively influence under-developed frontal brain regions involved in letter fluency-related skills [12], which in turn, might affect verbal ability. Moreover, based on results in adults, it is likely that CRP could alter academic performance through its negative effects on brain morphology and cognitive domains [28].

On the other hand, in the present study there was a lack of association between WBC and IL-6 with academic performance. Similarly, other previous interventional [29] and prospective [13] studies investigating the association between inflammation and cognitive function in youth have reported null findings. There are several hypotheses that could partially explain the lack of association found in our study. First, it is interesting to highlight that the lack of association found between IL-6 and academic performance indicators is generally consistent with previous research, suggesting that IL-6 does not affect proliferation and gliogenesis [4], which in turn, may have no influence on cognition or academic performance. Second, the fact that circulating inflammatory biomarkers at physiological levels can act with both, anti- and pro-inflammatory effects [10] could partially explain the divergent results found in the present study. Third, our sample showed optimal values of adherence to the Mediterranean diet and body composition, which have been related to lower levels of inflammatory biomarkers [21, 22]. Although in our sample levels of WBC and IL-6 are within the published range, they seem to be lower than the mean values reported in the existing literature [30, 31], which could partially explain that these concentrations did not affect students’ academic performance. Lastly, the academic performance indicators included in the current study might not entirely capture the adverse effects that some circulating inflammatory biomarkers could have on cognition in adolescents.

The mixed results found in prior literature investigating the relationship of inflammation with cognition and academic performance in youths could be due to differences in participants’ socioeconomic status [32], ethnicity [33] and lifestyles, as well as to methodological issues. In fact, divergent results could be related to the matrix (whole blood, serum or plasma, saliva) in which inflammatory biomarkers are measured in the studies [12], and even to the different technics of analysis implemented.

Limitations and strengths

The results of the present study should be interpreted with caution. The cross-sectional design of our study does not allow us to draw any conclusion on the causal direction of the associations. In addition, the inclusion of a sample of apparently healthy adolescents limits the generalizability of our findings across the population. Likewise, it is plausible that the present study includes residual confounding from unmeasured variables such as direct diagnosis of acute illnesses (e.g., respiratory, gastrointestinal, or dental problems) from a physician. This is important since acute disease conditions, which are related to increased inflammatory proteins, could have been underestimated by participants. This issue, together with the fact that inflammatory levels were not evaluated several times, may have influenced the associations reported. Finally, multiple testing could involve an increase of the type I error rate (i.e., false positive). However, the strengths of the study comprise the use of different blood-derived inflammatory biomarkers, and the inclusion of a standardized test of academic abilities. In addition, our statistical analyses were controlled for pubertal stage, socioeconomic status related variables [19], waist circumference [20, 21] and adherence to the Mediterranean diet [17, 22], which are relevant given their association with inflammation and academic performance as suggested by previous research.

Conclusions

In conclusion, our results suggested that some circulating inflammatory biomarkers were associated with academic performance in adolescents. Specifically, TNF-α was inversely associated with academic grades, while CRP was inversely associated with verbal ability. However, no association was found between WBC and IL-6 with adolescents’ academic performance. Additionally, our results indicated that inflammatory biomarkers could be useful to identify students with higher risk of low academic performance. Since academic performance has shown to play a key role on future employability [15] and health status [14], further larger longitudinal and interventional studies are needed to clarify the short-term and long-term relationship between inflammation and academic performance in youths.

Parameters of the receiver operating characteristic curve analysis for the diagnostic performance of circulating inflammatory biomarkers in identifying low academic abilities.

(DOCX)

Click here for additional data file.

20 Aug 2020

PONE-D-20-17331

CIRCULATING INFLAMMATORY BIOMARKERS AND ACADEMIC PERFORMANCE IN ADOLESCENTS: DADOS STUDY

PLOS ONE

Dear Dr. Moliner-Urdiales,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

What was the basis for considering sex, pubertal stage, parental educational level, type of school, waist circumference and adherence to the Mediterranean diet were included as covariates? Is there any evidence that variables like waist circumference and adherence to the Mediterranean diet affect the outcome of interest?

In the study economic status related variables have not been measured and adjusted.  At least theoretically, low household economic may lead to both of inflammation and low academic performance and confound the relationship of interest. Why it was not possible to measure and adjust such variables in the study?

Please state the statistical basis for reaching at the sample size of 244.

Results: I don’t see any sub-section that summarizes the findings of the logistic regression analysis. Please include a short paragraph or so.

Discussion: Please discuss limitation of the study including residual confounding from unmeasured variables and possible inflation of the type I error rate due to multiple testing.

Its good that only students free from chronic diseases were included in the study. But it is not clear how the health status of the students was assessed.

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Additional Editor Comments:

Table 1: Some of the significant differences between the two genders are not identified (e.g. academic scores for English and Spanish).

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Could you therefore please include the title page into the beginning of your manuscript file itself, listing all authors and affiliations?

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"The DADOS Study is funded by the Spanish Ministry of Economy and Competitiveness, MINECO (DEP2013-45515-R) and by the Jaume I University of Castellon, UJI (P1·1A2015-05; UJI-A2019-12).

This work is partly supported by a Sunny Sport research grant from the Schweppes Suntory Spain Company. MAR was supported by grant UJI E-2018-21. J.M was supported by grants FCT SFRH/BSAB/142983/2018 and UID/DTP/00617/2019, as well as Programa de Bolsas Santander Universidades 2018."

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors present a cross-sectional study of 244 boys and girls in order to test the hypothesis that inflammation mediators are associated with lower academic achievements.

The background information correctly argues for the feasibility of the hypothesis, and cite correct and up-to-date references.

Children were selected from both public and private schools, and all were on the same grade. The authors may wish to comment how many schools were approached and their possible representativeness in their community.

Little is shown about the actual recruiting process. The authors state the children were healthy and without chronic disease, they may wish to describe how this was evaluated. Furthermore, these mediators are also related to acute conditions; such as respiratory, gastrointestinal or dental problems, which may be more frequent in this age group. Some acute ailments may show seasonal variations during the year and it is possible to have several children affected at once. The authors may wish to comment on this.

To my knowledge inflammation mediators (IL-6 and TNF) were correctly assessed, CRP and leukocytes were also correctly measured (though, these are not mediators, but rather may show the effect of the mediators).

Academic performance was measured by both, grade point average on several subject maters and on a standarized test. It is important that the authors state if this test was applied at the same time of the measurement of mediators.

Covariates for the analysis were sex, pubertal development, parental educational achievement, type of school, and compliance with Mediterranean diet. Body mass index and waist circumference were also considered.

Logistic regression was performed with the value that best discriminated in ROC curve analysis; between achievement and the mediators. Since the values obtained with ROC curve analysis are close to the median of achievement (between 5 and 6); it would be important to know how these ratings relate to fail-pass grades in the Spanish educational system.

In the end TNF was associated to most of academic achievement ratings, and CRP was associated with verbal abilities ratings.

In the discussion, the authors explain their findings with previously published possible effects of both TNF and CRP in the central nervous system. Yet, the reasons for having higher mediators values are not fully explained. I believe that Mediterranean diet may not explain the full extent of these abnormalities. The authors may wish to comment on the degree of abnormality (i.e. compare these differences to normal values in their laboratory).

The greatest confounder in these circumstances tends to be obesity. ¿how many of their children were obese or overweight?, even if they used waist circumference in their logistic analysis.

The association makes perfect sense. Beyond the possible effects of TNF on the brain, If a child doesn’t feel well his/her performance will be substandard.

I believe the authors should comment on this aspects before the paper can be published.

Reviewer #2: - Please explain what method you have used to select your sample? e.g. randomly, convenience????

- Please explain how you select the schools.

Table 2. Please include 95% CI. You may exclude the none significant variables out of the table and explain those in the result?

Table 3. could you present table 3 in figure? may be the significant ones?

Minor comment: discussion - lines 211 - You may want to say the main finding of the present study reveals an association between TNF - alpha and math........ please fix similar wordings throughout.

Major comment.

We know that cognition as well as academic performance are results of various factors including nutrition, socio-economic factors, health status, genetic, environment etc or we can say long term effect of various factors. We also know that inflammation markers such as CRP and TNF alpha are early (maximum of 48 hrs) markers of inflammation or infection.

Now, my question is on the research question - are you trying to answer that inflammation is associated with cognitive and/or academic performance or you are tying to show that inflammation markers are associated with for instance, neurogenesis??? you have provided one in vitro-study that shows this relationship, is there any evidence from in vivo study that you could provide?

simple example. If the subjects were free of inflammation three days or more before data collection would the result be different?

Conclusion: should be based on your data.

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Reviewer #1: No

Reviewer #2: No

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5 Oct 2020

Dear Samson Gebremedhin,

My co-authors and I are pleased to respond the editor’s and the reviewers’ comments and resubmit a revised version of the paper entitled: “Circulating inflammatory biomarkers and academic performance in adolescents: DADOS study” submitted as a Research article to PLOS ONE (No. PONE-D-20-17331). Changes to the original manuscript appear in blue text for ease of identification, and we believe that our manuscript is stronger as result of these modifications. An itemized point-by-point response to the journal requirements and the reviewers’ comments is attached to the resubmitted manuscript.

Yours sincerely,

The authors

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

26 Oct 2020

CIRCULATING INFLAMMATORY BIOMARKERS AND ACADEMIC PERFORMANCE IN ADOLESCENTS: DADOS STUDY

PONE-D-20-17331R1

Dear Dr. Moliner-Urdiales,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Samson Gebremedhin, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

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Reviewer #1: No

Reviewer #2: No

29 Oct 2020

PONE-D-20-17331R1

Circulating inflammatory biomarkers and academic performance in adolescents: DADOS study

Dear Dr. Moliner-Urdiales:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Samson Gebremedhin

Academic Editor

PLOS ONE