When one's birthplace leads to advantages in sport participation or performance, it is called a birthplace effect. Nearly two decades of committed research has revealed that birthplace effects are pervasive across sports and countries. Recently, researchers have attempted to better understand birthplace effects by considering various metrics that serve as proxies for birth advantages; for instance, population size, population density, and proximity to sport clubs. Underlying mechanisms that explain birthplace effects include infrastructure  and social structure , though contextual differences across existing research  make it difficult to fully explain the effects. Herein, we provide more depth regarding these elements of birthplace effects, while also presenting new data on “talent hotspots”; that is, communities with optimal population  When considering the factors that influence elite talent development in sport, most would point to genetics, intrinsic motivation, passion, and work ethic. Perhaps fewer people might identify other contributing factors such as coaching and birthdate. Often overlooked, albeit quite important, is the influence that geography  has on talent development. A seminal study by Côté et al.  firmly pThe process to identify birthplace effects is relatively simple. First, a sample of athletes is selected for examination . Second, the athletes are placed into various categories, based on the population of their birthplaces . Next, the percentage of athletes in each category is calculated . Following, the percentage of the general population born in each population category is calculated . Finally, across each population category, the distribution of athletes is compared to the distribution of the general population . The mathematical calculations to identify if the distribution differences are “significant” or “meaningful” require more depth of understanding, which goes beyond the necessary description for this chapter. Suffice to say, these steps allow one to conclude if athletes born in a certain population category are (1) more, (2) less, or (3) no more likely to achieve elite sport status.Though one's birthplace is rather arbitrary, researchers have uncovered birthplace effects spanning countries and sports. Côté et al.  conducteIn the years following Côté et al.'s  study, tWhereas birthplace effects (as measured by population size) are fairly consistent, there is considerable variability in the advantaged population categories across existing studies. For instance, some studies show optimal birthplaces being communities of 50,000–99,999 residents , with ot2), yet handball players were more likely to be born in less-dense communities (100–250 residents/km2). Rossing et al. (2) produced more players. Similarly, van Nieuwstadt et al. (2) increased the likelihood of achieving the highest competitive divisions compared to athletes born in communities with higher population density (~400 residents/km2).Rossing et al.  exploredg et al.  continuet et al.  noted tht et al.  exploredThough population density might better reflect community structures, it still seems that results differ based on sport and country. As such, researchers have continued to employ other metrics to explore birthplace effects. These include considering proximity to the nearest soccer clubs , the coninfrastructure captured environmental structures available to youth athletes that might facilitate talent development. Examples include training facilities, competition venues, available parks and green spaces for unstructured play, and teams or clubs that focus on development rather than winning. Of note, training and competition venues need not be state-of-the-art (often the case in large cities); rather, the venues merely need to be nearby and available for frequent use. Second, social structure centers on family and community factors that yield positive sport outcomes. The authors posited that social structure includes autonomy-supportive coaches, involved (but not over-involved) and caring parents, environments where unsupervised youth feel safe, and sport programs that promote positive youth development. The notion that optimal social structures yield favorable birthplace effects was previously intimated by Hancock and Côté , though we moved to a different city  before he was 6-weeks-old. If asked again in 10 years, he might state the latter city as his birthplace, likening it more to a “home town.” Since the populations of these two cities are quite different, his response to that question could lead to different interpretations—similar to what one would expect of athlete populations when asked the same question. Not only is birthplace self-reported, but in most instances, data are collected by sport organizations, not researchers. Since there would rarely be a need for sport organizations to differentiate between birthplace and community of development, it likely means that little guidance is given to athletes about what constitutes birthplace. The nature of birthplace as defined and measured in the literature has several limitations that contribute to our lack of understanding of birthplace effects. Researchers would be wise to acknowledge these issues, while also seeking data collection techniques that rely on direct information from participants rather than archival methods.A second concern relates to the community sizes that constitute each population category in birthplace effects research. Across the globe, many municipal governments have amalgamated to form larger cities with fewer administrative costs and streamlined—or at least centralized—community services. This raises a concern for birthplace effects researchers, who must deliberate on the population categorizations of amalgamated cities. Going back to the first author's son, his actual birthplace was a suburb of the larger city, which had a population of ~25,000. This brings into question what his assigned birthplace population size should be: 25,000 (the size of the suburb) or 1,000,000 ? Typically, more accurate results come from specifying suburbs and amalgamated regions, though when reliant on sport organizations to provide birthplace data, researchers must accept whatever data were collected, even if they are not ideal. Again, this points to the need for researchers in this field to consider direct data collection measures, along with clear decision processes for categorizing various community sizes, to improve the validity of their findings.Recent research overseen by Wattie , 18, 19 A final weakness related to birthplace effects is the type of research that is typically conducted. Most often, researchers employ archival methods  to explore birthplace effects. Such approaches have been vital in identifying the presence of birthplace effects across sports and countries, but they have been less useful for contributing to our understanding of why birthplace effects exist. Instead, researchers ought to seek varied methods for future research including direct participant interactions (as noted above), longitudinal or quasi-longitudinal designs (to track changes over time and shifts in the communities in which athletes live), and qualitative methods .The fact that this section is longer than the preceding ones speaks volumes to how limited our knowledge is of birthplace effects. This is partially because it is a field still in its infancy, but also because several weaknesses in researchers' approaches render it challenging to draw firm conclusions about the field. Hopefully those reading this chapter (students or researchers) take it as a call to action and are inspired to create research designs with the goal of standardizing birthplace effects measures/metrics that lead to meaningful conclusions.The following section aims to draw further attention to some of the gaps in the birthplace effect literature identified above. In response to critiques challenging the homogeneity of communities of particular sizes , 19, we n = 382), Women's National Basketball Association , Men's Division I National Collegiate Athletic Association , and Women's Division I National Collegiate Athletic Association  using each team's 2018 rosters.A total of 8,740 American professional and collegiate basketball players were included in the study. Places of birth were collected from the official websites of the National Basketball Association  50–99 residents/km2, (3) 100–249 residents/km2, (4) 250–499 residents/km2, (5) 500–999 residents/km2, (6) 1,000–2,499 residents/km2, (7) 2,500- 4,999 residents/km2, and (8) ≥5,000 residents/km2.Communities were categorized into eight groups based on population size: (1) <50,000 residents, (2) 50,000–99,999 residents, (3) 100,000–249,999 residents, (4) 250,000–499,999 residents, (5) 500,000–999,999 residents, (6) 1,000,000–2,499,999 residents, (7) 2,500,000–4,999,999 residents, and (8) ≥5,000,000 residents. For population density, eight groups were used: (1) <50 residents/kmOdds ratios (ORs) were calculated across the different population sizes and densities for each group. ORs and 95% confidence intervals (CIs) were calculated by dividing the chance of being a part of the sample group from a population size or density  by the chance of being from a specific community with that population size or density based on the general population . ORs were interpreted based on their positioning above or below 1. An OR above 1 implies that an athlete in that population size or density has a greater likelihood of participating at elite levels compared to an individual from a different population size or density. Conversely, ORs less than 1 means that an athlete is less likely to achieve success from a certain population size or density than someone from another area. If the CI contains the 1, those ORs are deemed not statistically significant.MNCAA = 1.94, CIMNCAA = 1.75–2.14; ORWNCAA = 1.79, CIWNCAA = 1.62–1.98). While the specific ORs differed slightly across male and female NCAA athletes, the overall trends in relation to community size were consistent across genders.There was a significant over-representation of both male and female NCAA athletes from two community size categories, ranging from 100,000 to 499,999 residents . This ef2 . Similar to the community size data, ORs followed similar trends across density categories in both male and female NCAA athletes.As for population density, there was a significant over-representation of male and female collegiate athletes from moderately dense cities between 500 and 2,499 residents/km2 , with thNBA = 2.90, CINBA = 2.20–3.81), while the WNBA showed the largest over-representation in the 500,000–999,999 residents category . Besides the ranges noted above, NBA athletes were also significantly over-represented from cities between 50,000 and 99,999 residents  and 2,500,000–4,999,999 residents .Among both NBA and WNBA datasets, there was a significant over-representation of male and female participants from three community size categories ranging from 100,000 to 999,999 residents see . For the2) were 8.3 times more likely to produce professional basketball players . For the NBA, the only population density category that was significantly over-represented was 2,500–4,999 residents/km2 , which was also over-represented in the WNBA sample .As for population density, there was an over-representation for both the NBA and WNBA in relatively dense cities . For the2), while WNBA athletes were highly over-represented in extremely dense population centers .The purpose of this study was to analyze the birthplace effects in both collegiate and professional United States basketball based on population size and density. By collecting data from two competitive levels and sexes, we were able to extend upon extant literature by providing a more nuanced understanding of birthplace effects in the sport of basketball in the United States. Notably, the largest over-representation in community size for NBA athletes were from cities between 250,000 and 499,999 residents, while WNBA athletes were most over-represented from cities between 500,000 and 999,999 residents. For both collegiate males and females, the highest representation was from cities between 250,000 and 499,999 residents. For population density, NBA athletes were most likely to be from moderately dense areas . Similar findings were observed for collegiate athletes, who additionally were found to be heavily over-represented from medium density communities (2). Altogether, these findings were consistent with prior birthplace effect studies that have considered density in European populations  professionals .2). In women's basketball, there was a significant over-representation of both collegiate and professional players from communities with populations of 100,000–499,999, with the data skewing toward increasingly dense communities in the transition from the collegiate  to professional ranks . Finally, in examination of collegiate and professional men's and women's basketball altogether, it is evident that communities of 100,000–249,999 residents can be considered “talent hotspots” as there are significant over-representations of athletes from communities of this size across all four data sets.One of the unique objectives of this study was to identify potential “talent hotspots” in both men's and women's basketball in the US. Through the analysis of the optimal community sizes and densities at both the collegiate  and professional ranks, it was possible to identify community sizes and densities that are over-represented at both levels of competition. In men's basketball, there was a significant over-representation of collegiate and professional players from communities with populations of 100,000–999,999 residents. In terms of population density, there were no significant overlapping ORs across the men's collegiate and professional datasets. However, both data sets favor relatively dense communities  and social structure (related to density) impact athlete development, to begin exploring other indices of these communities that may be optimal for the development of elite basketball players .It is evident that geography is a significant factor influencing talent development among athletes. Likely, this is because of favorable infrastructure and social structure that exist in certain communities. As indicated by the data presented herein, communities with optimal population sizes and densities might be considered talent hotspots. However, researchers ought to continue exploring other elements within such communities  to deepen our understanding of these potential hotspots. Such explorations might also identify environmental/geographic factors that consistently explain talent development advantages across sports and countries.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.DH led the writing of most sections of the paper. MV led the writing of the final section which includes the original research. AN contributed with article reviews, referencing, and reviewing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nonalcoholic fatty liver disease (NAFLD) is one of the most common health problems worldwide. Sleep apnea (SA) causes cardiovascular and metabolic problems, as well as a significant socioeconomic burden. Although several studies have found that SA causes NAFLD, there is no evidence that NAFLD causes SA. The goal of this study was to look at the relationship between NAFLD and SA in realworld data.We evaluated 334,334 healthy individuals without comorbidities who underwent National Health checkups in the Republic of Korea from 2009 to 2014. NAFLD was defined by a surrogate marker, the fatty liver index (FLI). The association between FLI and SA was analyzed using multivariate Cox proportional hazards regression models.P < 0.001). Adjusted hazard ratios (HRs) revealed that a higher FLI was independently associated with an increased risk of SA . This association remained statistically significant after further adjustment for Body mass index (BMI) . FLI was significantly associated with an increased risk of new-onset SA regardless of baseline characteristics.During a median followup of 5.3 years, 1,351 patients (0.4%) were newly diagnosed with SA. Subjects were categorized into quartile groups according to FLI . Subjects with higher FLIs had a significantly higher cumulative incidence of SA than those with lower FLIs (Q1, 119 [0.1%]; Q2, 210 [0.3%]; Q3, 339 [0.4%]; Q4, 683 [0.8%]; This study demonstrated that NAFLD, assessed by FLI, was independently associated with increased risk for SA in the healthy Korean population. Nonalcoholic fatty liver disease (NAFLD) is one of the most common health problems worldwide. Because NAFLD is typically a silent disease, exact incidence rates are unknown; however, it is commonly estimated to be in the range of 27%–34% in the general population . ObesityAlthough several studies are showing a positive association between SA and NAFLD using fatty liver index (FLI) or histologic examination, they are small sample sizes and showed a simple association in several cross-sectional studies , 9. PrevWe analyzed the National Health Insurance Service–National Sample Cohort 2.0 (NHIS-NSC 2.0) data set which is the population-based sample cohort data of Korea. The NHIS-NSC 2.0 was made by extracting random samples of about 2% of the total Korean population in 2006 with retrospective and prospective followup data from 2002 to 2015. Koreans are required by law to obtain government-provided National Health insurance, and NHIS covers 97% of Koreans. The Korean government stratified insured population  into 2,142 classes according to their by gender, age, region, eligibility status, and income level. The Korean government then randomly selected 2.1% from each stratum . As a result, the NHIS-NSC 2.0 cohort can represent the entire Korean population .The cohort contains four databases: the first is a sociodemographic information dataset of the beneficiaries; the second is a medical claims dataset with diagnoses based on the 10th revision of the International Classification of Disease (ICD-10) codes, admission, and treatment; the third is a National Health Screening dataset of the cohort members; and the fourth is a medical institutions dataset.Korean adults over the age of 20 are recommended the National Health checkup biennially, which is composed of blood tests, chest X-rays, physical examinations, and questionnaires on medical history and health-related behaviors including smoking status and alcohol consumption. According to NHIS data from 2013, 72.1% of eligible beneficiaries received a National Health checkup [All researchers whose protocols have been approved by the NIHS review board can use the NHIS-NSC 2.0. The Chungnam National University Hospital’s Institutional Review Board in Daejeon, Korea, approved this study and waived the requirement for informed consent.This study included a population over 20 years of age who received National Health checkups at least once from 2009 to 2014. The index checkup was defined as the first National checkup, and the index year was the year of the index checkup. Subjects who met the predefined exclusion criterion were excluded.: (a) those suffering from congestive heart failure, hypertension, diabetes, cerebrovascular disease, ischemic heart disease, peripheral artery disease, or liver disease; (b) those with rheumatic or nonrheumatic valvular heart disease; (c) those who with increased baseline SBP ≥140 mmHg or DBP ≥90 mmHg, and fasting blood glucose ≥126 mg/dL at the index checkups; and (d) those found to have missing data in the index checkup. Each diagnosis was based on 1-year clams data before the index year or index checkups . We defined each diagnosis in the claims data as the first occurrence on at least two different days of hospital visits (outpatient) or on the first admission. The ICD-10 codes are used to define each diagnosis.2). The physical activity questionnaires included questions about how many days a week 30 minutes of light exercise, 30 minutes of moderate exercise, and 20 minutes of vigorous exercise are performed, respectively. The light exercise was assumed to be 2 metabolic equivalent of tasks (METs), moderate 3METs, and vigorous 6METs, which were multiplied by 20, 30, and 30 minutes and the respective number of days a week, and summed.Smoking status was categorized into ex- or current smokers, and nonsmokers. Alcohol consumption was assessed using standardized self-reporting questionnaires that included questions about how many days of the week alcohol is consumed and how much alcohol is consumed on each drinking day. The amount of alcohol consumption was calculated by multiplying them. Body mass index (BMI) was calculated by dividing weight (kg) by height squared , or study completion .A well-validated, surrogate marker, FLI was used to identify patients with steatosis . FLI wasThe original study suggested an FLI ≥ 60 as the cutoff for a diagnosis of hepatic steatosis with a positive likelihood ratio of 4.3 in the general population . AlthougData are presented as mean ± standard deviation for continuous variables and as number (percentage) for categorical variables. The differences between FLI quartiles were compared using oneway analysis of variance and chi-square tests. The incidence of SA was calculated by dividing the total number of newly diagnosed SA cases between 2009 and 2014 by the total population during the same period and expressing the result as cases per 100,000 person-years. Cumulative event rates were calculated and compared between FLI quartile-based groups using Kaplan–Meier estimates and the log-rank test. For SA incidence, Cox proportional hazards regression was used to estimating adjusted hazard ratios (HRs), and 95 percent confidence intervals (CIs) were used. Data were first adjusted for age and gender (model 1). Then, clinical characteristics of which association with new-onset SA demonstrates borderline statistical significance (P < 0.10), smoking, alcohol consumption, activity, systolic blood pressure, diastolic blood pressure, fasting glucose, and low-density lipoprotein cholesterol were incorporated into model 1 to obtain model 2. In model 3, multivariate analysis was performed, and BMI, which is an important risk factor for both NAFLD and SA, was added to model 2. The variance inflation factor was used to test the multicollinearity of the variables included in the analysis, and all of them were less than 10, indicating that they had no effect on the results.P value of <0.05, was considered to indicate statistical significance. R software, version 3.3.3, was used for statistical analyses .For subgroup analyses, FLI was integrated into the statistical models as a continuous variable after log transformation. A two-sided Of the total 556,884 NHIS-NSC 2.0 sample cohort population, the exclusion of subjects who met the prespecified exclusion criteria left 334,334 subjects into the analysis. P < 0.10 in the univariate analyses (model 2), the HR (95% CI) was 4.03 (3.22–5.05) for the highest group of FLI compared with that of the lowest. The relationship between FLI quartile and SA incidence remained statistically significant after adjusting for BMI in model 2 , during which 1,351 subjects (0.4%) developed new-onset SA.  model 2 .In addition to the FLI groups, the analysis was carried out after categorizing the study subjects according to the various FLI cutoff criteria proposed in previous studies. Regardless of the cutoff-points used, the highest FLI goup had the highest risk for new onset SA.Adjusted HRs according to subgroups summarized in In this study, we looked at the relationship between NAFLD as measured by FLI, a well-validated surrogate biomarker, and the prevalence of SA in a large, realworld data set from the Korean population. Recent studies demonstrate an association between NAFLD and SA , 14, 15.Several mechanisms potentially link NAFLD and SA beyond the shared risk factors. Firstly, the wider neck circumference in NAFLD patients than in the general population may cause SA. NAFLD patients have a wider neck circumference (NC) than healthy subjects, according to previous research . Kim et Given the observed link between NAFLD and SA, as well as the mechanisms underlying this link, it is reasonable to assume that interventions for NAFLD management may have the therapeutic potential to reverse the natural course of SA. Obesity is widely recognized as a major risk factor for SA, and if achieved, significant and sustained weight loss is likely to be a useful therapeutic option in the management of SA . BecauseSeveral limitations of this study need to be acknowledged. To begin, we estimated new-onset SA using the NHIS-NSC 2.0 database, which is based on ICD-10 disease codes; however, the accuracy of SA diagnosis was not validated in this study. More research is needed to validate SA patient data with health insurance data using hospital-based medical records. Second, steatosis was determined using a surrogate biochemical marker rather than histology or magnetic resonance spectroscopy. An invasive procedure or a complex, expensive imaging modality, on the other hand, is not feasible in large cohorts. FLI, on the other hand, is a widely accepted surrogate for hepatic steatosis that has been validated in the general population . InteresIn conclusion, our study demonstrated that NAFLD, as measured by FLI, was independently associated with increased risk for SA in the healthy Korean population. Because management of SA remains a difficult clinical challenge, the identification of potentially modifiable risk factors may have important for clinicians. More research on the effect of NAFLD management on the incidence and severity of SA is required.


To compare pulmonary function parameters and the prevalence of altered pulmonary function in children born preterm and full-term, using the Global Lung Initiative reference values.American Thoracic Society and European Respiratory Society Technical Statement. The inclusion criteria were, among the preterm group: gestational age <37 weeks and birth weight <2000g; among the full-term group: schoolchildren born full-term with birth weight >2500g, recruited at two public schools in São Paulo, Brazil, matched by sex and age with the preterm group. As exclusion criteria, congenital malformations, cognitive deficit, and respiratory problems in the past 15 days were considered.This is a cross-sectional study with 6–9-year-old children submitted to measurement of airway resistance (Rint) and spirometry according to the 1)/FVC , FEV1 , and forced expiratory flow between 25% and 75% of vital capacity (FEF25-75) , respectively. The prevalence values of altered airway resistance  and spirometry  were higher in preterm infants than in full-term ones.A total of 112 children were included in each group. Preterm children had gestational age of 30.8±2.8 weeks and birth weight of 1349±334g. Among them, 46.6% were boys, 46.4% presented respiratory distress syndrome, 19.6% bronchopulmonary dysplasia, and 65.2% were submitted to mechanical ventilation in the neonatal unit. At study entry, both groups were similar in age and anthropometric parameters. Parameters of pulmonary function (Z scores) in preterm and full-term groups were: Rint ; forced vital capacity (FVC) , forced expiratory volume in one second (FEV1/FVC and FEF25-75 compared with those born full-term.Preterm children had higher prevalence of altered pulmonary function, higher Z scores of airway resistance, and lower Z scores of FEV Comparatively, Gonçalves et al., using the Polgar and Promadhat’s curve, observed a much higher prevalence of altered pulmonary function in children and adolescents born with a mean of 32 weeks of gestational age, reflecting the influence of the curve used in the assessment of pulmonary function.Thus, Thunqvist et al.In this context, the present study aimed to compare the prevalence of altered pulmonary function and to analyze pulmonary function parameters in schoolchildren born preterm and full-term, using the GLI curve as reference.This is a cross-sectional study with two groups of schoolchildren aged 6 to 9 years, one group of preterm infants and a control group of full-term infants, submitted to the pulmonary function test, carried out from December 6, 2016 to March 8, 2019.The preterm group included children with gestational age <37 weeks at birth and weight <2000g, born in the neonatal units of Universidade Federal de São Paulo (Unifesp), in São Paulo, Brazil, and selected from the follow-up list of the Premature Outpatient Clinic of the aforementioned institution. After discharge from the neonatal unit, preterm infants are routinely referred to the Premature Outpatient Clinic, where they are followed up until the end of adolescence. During the follow-up period, they are regularly assisted by a team of pediatricians, neuropediatricians, physiotherapists, speech therapists, ophthalmologists, psychologists, psychopedagogues, dentists, nutritionists, and social workers.The control group included children of the same age, born full-term, weighing >2500g, matched by age and sex with the preterm group and recruited from two public schools in the city of São Paulo, according to the researcher’s convenience.Initially, the list of children born preterm who met the inclusion criteria was compiled. Once having this list, the authors selected the first schoolchildren on the attendance list that could be matched by age and sex with each child in the preterm group.In both groups, children with congenital malformations, neuromuscular disorders, and cognitive deficits that limited the understanding of the guidelines for performing pulmonary function were excluded, in addition to those with respiratory problems, such as respiratory infections, wheezing, or seeking emergency room for symptoms less than 15 days before the test.. The International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire was used to diagnose childhood asthma.The clinical history regarding the birth and follow-up of preterm infants was collected from the medical record and supplemented with interviews with the children’s parents. For the full-term group, a questionnaire was sent to be completed by the parents. The Fenton and Kim’s curve was used to classify the neonatal populationDemographic and clinical data collection and pulmonary function tests were performed on the same day for each child included in the study.Parents/guardians of the children were instructed to discontinue medications known to influence bronchial responsiveness 24–48 hours before the test, as recommended worldwide.® portable equipment  was used following the international recommendations of the American Thoracic Society and the European Respiratory Society Technical Statement (ATS/ERS). The tests took place with children comfortably seated, using a 3cm-diameter disposable mouthpiece and nose clip.All pulmonary function measurements were taken by the same researcher, using the interrupter technique (Rint) to measure airway resistance and spirometry measures. The ROCC Pony FX the flow was measured during breathing at tidal volume, with the shutter automatically closed after 10ms of the peak expiratory flow, remaining closed until 100ms. Pressure was measured using two points in the linear back-extrapolation method. Seven airflow interruptions were performed at the unforced peak expiratory flow, with random and automatic frequencies. The measurement was considered successful if at least five appropriate measures were obtained as well as the coefficient of variation of the measures ≤20%.In the interrupter technique,Measures in which air escaped through the mouthpiece, excessive neck extension or flexion, irregular breathing pattern, movement during closure of the shutter, coughing, swallowing, or sneezing were excluded., the flow/volume and volume/time curves were analyzed and excluded if they were visibly inadequate according to the reference standard. The allowed extrapolated volume was <15mL or <5% of the FVC (whichever was greater). The test ended when the volume/time curve reached the plateau and the child was unable to continue exhaling, that is, there was no change in volume per second. Reproducibility was assessed using two acceptable curves in which the second highest FVC and FEV1 values were <150mL.For spirometry measures®), and four puffs were sprayed to reach the recommended dose of medication. Subsequently, pulmonary function tests were repeated after 15 minutes of bronchodilator inhalation.After spirometry measurements, the children were submitted to inhalation with aerosol with bronchodilator  using a spacer device . Parents were requested to sign an Informed Consent Form, and children were requested to sign the Assent Form. considering the 32% prevalence of altered pulmonary function in schoolchildren born preterm, and on the study by Kilbride et al., that showed a prevalence of 12% in full-term children. Considering an alpha error of 5%, beta error of 80%, and loss of 20% due to possible problems in the acceptability and reproducibility of pulmonary function curves, the need to include 113 children in each group was noted.The sample size calculation was based on the study conducted by Gonçalves et al.,t-test  or Mann-Whitney’s test . The assessment of the normality of distribution was verified by the Kolmogorov-Smirnov test. Categorical variables were expressed as number and percentage, and were compared by Chi-square or Fisher’s exact test. Statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS) program, version 17 , considering p<0.05 significant.Numerical variables were expressed as mean and standard deviation and were compared by Student’s A total of 114 children aged 6–9 years in the preterm group and 115 in the full-term group met the inclusion criteria. Of these, two children born preterm and three born full-term were unable to perform spirometry. Thus, in each group, 112 children were studied, and 52 (46.4%) of them were boys. The mean birth weight was 1349±334g and 3182±453g, respectively, in the preterm and full-term groups (p<0.001).Mothers of preterm children were 29.6±7.3 years old at the time of delivery. During pregnancy, 39 (34.8%) had gestational hypertension; 12 (10.7%), diabetes mellitus; eight (7.1%), hypertension; 30 (26.8%), urinary tract infection; one (0.9%), chorioamnionitis; three (2.7%), placenta previa; and two (1.8%), placental abruption. Antenatal corticosteroid was administered in 62 (55.4%) pregnant women.The mean gestational age of the preterm group was 30.8±2.8 weeks, and 40 (35.7%) newborns were small for gestational age. During hospitalization in the neonatal unit, 52 (46.4%) preterm infants presented respiratory distress syndrome; 17 (15.2%), patent ductus arteriosus; 23 (20.7%), neonatal sepsis; six (5.4%), meningitis; 37 (33%), periventricular and intraventricular hemorrhage; 19 (17%), retinopathy of prematurity; and 22 (19.6%), oxygen dependency at 36 weeks of corrected age.In the neonatal unit, 73 (65.2%) preterm infants required invasive mechanical ventilation with a mean ventilation time of 3.2±8.4 days . Hospitalization time was 45.3±26.8 days . the presence of wheezing after physical exercise, and the need for hospitalization were higher among premature infants than among those born full-term. In all, 44 preterm infants were hospitalized at least once during childhood. Most hospitalizations were due to respiratory disorders, with 16 hospitalizations due to pneumonia; 16 due to bronchiolitis; three due to asthma; two due to influenza; and the others due to meningitis, seizures, or hernia or phimosis surgeries  and spirometry  was higher in the preterm group compared with the full-term group.Among the children born preterm, nine (30%) presented mild airway restriction; 11 (36.7%), mild airway obstruction; five (16.7%), moderate obstruction; one (3.3%), severe obstruction; and four (13.3%), mixed symptoms of airway restriction and moderate/severe obstruction. Among those born full-term, six (40%) presented mild airway restriction; eight (53.3%), mild airway obstruction; and only one (6.7%), moderate mixed condition (p<0.012).1, the positive response was found in 12 (10.7%) children born preterm and in six (5.4%) children born full-term (p=0.198).Regarding airway resistance measures, there was a positive bronchodilator response in 27 (24.1%) and 18 (16.1%) children in the preterm and full-term groups, respectively (p=0.133). For measures of FEVAmong schoolchildren born preterm, those with altered spirometry, compared with those with normal parameters, presented lower birth weight, higher frequency of oxygen dependency at 36 weeks of corrected age and, during childhood, a higher occurrence of pneumonia and asthma diagnosis and a tendency to higher BMI Z scores .1/FVC, FEV1, and FEF25-75, characterizing pulmonary impairment predominantly of an obstructive nature. Such results have been widely reported in the literature, reinforcing the hypothesis that prematurity has an impact on pulmonary function that may persist into adulthood.The present study showed that the preterm group, when compared with the full-term group, presented a higher prevalence of altered pulmonary function with higher airway resistance values and lower parameters of FEV similar to what was observed in the present study.However, it is worth mentioning that the results of studies on pulmonary function testing have varied according to the characteristics of the analyzed neonatal population. In addition to the degree of prematurity, other factors, such as bronchopulmonary dysplasia and mechanical ventilation, have been associated with greater pulmonary impairment, Based on the GLI curves adopted in this study, the prevalence of altered spirometry was 26.8% in schoolchildren born preterm, lower than the 43% found in another study on preterm infants with similar gestational age when adopting the reference curve of Polgar and Promadhat. Likewise, Fawke et al. reported a prevalence of 56% in schoolchildren born extremely preterm considering the reference values recommended by Stanojevic et al.Another variable that may contribute to differences in spirometry results in preterm infants is the choice of the reference standard for interpreting test results. described, according to GLI standards, a prevalence of alteration in 14% of 6.5-year-old children born with gestational age <27 weeks for FVC and in 23% of them for FEV1, evidencing the influence of the reference standard considered, among other possible factors.Conversely, Thunqvist et al.In the present study, the higher occurrence of respiratory diseases and wheezing in the past 12 months in the preterm group compared with the full-term group is noteworthy. Furthermore, the authors observed that children born preterm with altered pulmonary function had a higher incidence of childhood pneumonia and wheezing in the past 12 months. who reported a 40% prevalence of asthma-like disorder in extremely preterm infants compared with 15% of those born full-term at 6.5 years of age. Moreover, preterm infants who reported asthma had lower FEV1 Z scores compared with those without such history. These respiratory complications associated with prematurity, whether or not accompanied by a history of bronchopulmonary dysplasia, have been described quite frequently and may explain the predominance of obstructive disorders in individuals born preterm.These findings were also described by Thunqvist et al., also found a predominance of the obstructive pattern in pulmonary function tests in 224 adults aged 26 to 30 years born in the 1986 New Zealand cohort with a gestational age of 28.2±2.5 weeks, compared with 100 full-term controls. In the aforementioned study, a greater severity of the obstructive condition was reported in preterm infants with a history of bronchopulmonary dysplasia and a higher occurrence of asthma among preterm infants compared with those born full-term , similar to what was observed in the present study and others.Yang et al. A systematic review and subsequent meta-analysis of 16 studies showed that, among other factors, prematurity, low birth weight, pulmonary infections, and asthma in childhood are determinants of chronic obstructive pulmonary disease in adults.An important clinical implication is that the obstructive pattern may persist into adulthood and clinically manifest as a chronic respiratory disease. possibly due to lung growth with increasing age. Corroborating this result, it is noteworthy that the anthropometric parameters for inclusion in the study were, on average, adequate for age and similar in both groups, justifying the small difference between the prevalence of restrictive disorders among them. Nevertheless, it is worth emphasizing that in the preterm group the percentage of overweight/obese children was similar to the full-term group and also to that of the Brazilian population for the age group from 5 to 9 years old.In the present study, the prevalence of restrictive changes detected by spirometry was lower than the prevalence of obstructive ones, a result similar to that observed by other researchers, One possible explanation is that the multidisciplinary follow-up performed at the Premature Outpatient Clinic, with clinical and social support until inclusion in the study, would have contributed to reduce the difference in growth between the groups; however, a need for surveillance of excessive weight gain is observed, as there was a tendency for higher BMI-for-age Z scores among preterm infants with altered pulmonary function, confirming data from the literature that indicate changes in pulmonary function in obese individuals.This growth profile was also observed in another study. are considered the international gold standard, because they offer a unified approach for the interpretation of pulmonary function tests, in addition to being multiethnic and validated in many countries, including Brazil. Similarly, the inclusion of a control group of full-term schoolchildren and the sample calculation valorize the obtained results.This study presents a differentiated peculiarity concerning the reference standards used for interpreting pulmonary function tests. The adopted GLI curvesConversely, the cross-sectional design of the study, the convenience sample, and the difficulty accessing some data from the full-term group constitute limitations of the study. it is still a high prevalence for a sample of preterm infants with a mean gestational age of 31 weeks. In addition, the finding of higher airway resistance, lower values of FEV1/FVC, FEF25-75, as well as % of expected FEV1, higher prevalence of asthma, and significant proportion of children with positive bronchodilator response among preterm infants suggest the possibility that some of the studied children present a chronic respiratory disease resulting from prematurity.Finally, it is worth noting that, although the new reference standard showed a lower prevalence of pulmonary function alteration than that found in previous studies,All in all, this study showed that children aged 6–9 years old who were born preterm assessed by GLI curves showed a higher prevalence of altered pulmonary function, especially those of an obstructive nature, with higher airway resistance and lower spirometry parameters, compared with those born full-term.


Hand, foot, and mouth disease (HFMD) is a viral infection caused by a virus from the enterovirus genus of picornavirus family that majorly affects children. Though most cases of HFMD do not cause major problems, the outbreaks of Enterovirus 71 (EV71) can produce a high risk of neurological sequelae, including meningoencephalitis, lung difficulties, and mortality. In Asia, HFMD caused by EV71 has emerged as an acutely infectious disease of highly pathogenic potential, which demands the attention of the international medical community.Some online databases including NCBI, PubMed, Google Scholar, ProQuest, Scopus, and EBSCO were also accessed using keywords relating to the topic for data mining. The paid articles were accessed through the Centre Library facility of Siksha O Anusandhan University. This work describes the structure, outbreak, molecular epidemiology of Enterovirus 71 along with different EV71 vaccines. Many vaccines have been developed such as inactivated whole-virus live attenuated, subviral particles, and DNA vaccines to cure the patients. In Asia–Pacific nations, inactivated EV71 vaccination still confronts considerable obstacles in terms of vaccine standardization, registration, price, and harmonization of pathogen surveillance and measurements.HFMD has emerged as a severe health hazard in Asia–Pacific countries in recent decades. In Mainland China and other countries with high HFMD prevalence, the inactivated EV71 vaccination will be a vital tool in safeguarding children's health. When creating inactivated EV71 vaccines, Mainland China ensured maintaining high standards of vaccine quality. The Phase III clinical studies were used to confirm the safety and effectiveness of vaccinations. The EV71 virus generally causes hand, foot, and mouth diseases (HFMDs) or herpangina and neurological abnormalities among children below 5 to7 years old. It causes severe rashes in the hand, foot, and mouth that appear like blisters , 3. MoreHFMD causes severe health hazards such as abnormality in CNS, respiratory problems, cardiovascular disorders, aseptic meningitis, cerebella ataxia, poliomyelitis-like paralysis acute brainstem encephalitis, cardiopulmonary failure, fulminant neurogenic pulmonary oedema, and also increase in death rates . HFMD isHFMD can occur more by EV71 but less by coxsackieviruses as A16 (CVA16), A5, A6, A7, A9, A10, B1, B2, B3, and B5 . EnterovSolomon et al.  reportedA basic and thorough overview of the literature surveyed to identify the viral infection of EV71 was conducted till 2021. Many offline and online databases were taken into consideration. The review articles and research papers published by various reputed publishers such as Elsevier, Springer, Taylor & Francis imprints, and Hindawi were considered as the primary source of data collection for this review article. Some online databases including NCBI, PubMed, Google Scholar, ProQuest, Scopus, and EBSCO were also accessed using keywords relating to the topic for data mining. The paid articles were accessed through the Centre Library facility of Siksha O Anusandhan University. The conference proceedings, magazines, WebPages, and book chapters were also reviewed and accessed as the other sources of the literature to maximize the information about the current bottlenecks, the extent of research carried out, and the potential utility of the topic. In this review, it is discussed about the structure, outbreak, and molecular epidemiology of Enterovirus 71 along with different EV71 vaccines. The EV71 infection is a major threat to public health across the world. The infections are mostly seen in children and very rarely in adults.The structure of Enterovirus 71 is a single-stranded RNA virus with an RNA genome size of around 7.4 kb . FolegatMoreover, the capsid of EV71 has 60 copies of VP1, VP2, VP3, VP4, and the structural proteins are combined to make a protomer. Further, 5 protomers form a pentamer and 12 pentamers are combined to make a virion , 17. In Further, the Picornaviridae viral genomes are composed of IRES. So, the virus can enter the host cells and release the viral genome in the cytoplasm, and IRES can be interpreted by viral RNA. At the time of viral translation and replication, the virus needs IRES-specific trans-acting factors (ITAFs). It requires some other factors like T cell-restricted intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR) for viral translation and replication. The interaction of TIA-1 and TIAR with the 5´ untranslated region of the viral genome can increase the replication . The strEnteroviruses are classified into several types as poliovirus (PV) is a micro, unenveloped virus of the genus Enterovirus, with a single-stranded genomic positive ribonucleic acid of approx. 7500nt Picornaviridae family. The PV is a causal factor in poliomyelitis, where motor neurons are the primary target. The PV motor neuron tropism is due in part to PVR (PV receptor) expression. In this regard, EV71 relates to the EV community, but has been split as PV, human EV genus A, into many genera. In addition to, Enterovirus 68 has induced lower lung infections in the younger forms of Enterovirus 70, that is an agent of extreme acute haemorrhagic conjunctivitis epidemics, where EV71 (Enterovirus 71) has exacerbated aseptic meningitis and hand, foot, and mouth disease (HFMD) and encephalitis in a variety of nations. Aseptic meningitis, herpangina, outbreak myalgia , hand, foot, and mouth illness, myocarditis, pericarditis, diarrhoea, rashes, and sinus infections are signs with several diseases. In specific congenital malformations and maybe in some instances of diabetes, they can also play a part. Among all, the echovirus-induced diseases are aseptic meningitis, febrile infections without or with a cough, and rash. Table Some important EV71 viral receptors like P-selectin glycoprotein ligand 1 (PSGL-1), human scavenger receptor hSCARB2), human dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN), annexin A2 (AnxA2), heparan sulphate (HS), and sialylated glycan have the ability to bind with the host cells . After b, human dInitially, HFMD was detected in April 1957 with 8 children infected in New Zealand and during the 1960s the major outbreak of HFMD occurred by the CVA16 virus . In 1969In 1981, HFMD was first identified in Shanghai, China. Later, in 2008, approximately 488,955 cases were detected with a mortality rate of 126 in China , 26. BetMost EV71 cases detected in 1998 were with genotype C, while 1/10 was identified with genotype B. The death rate of EV71 was 78 in 1998, 25 in 2000, and 26 in 2001. Lin and Tzou categorized 1998 outbreak of Taiwan into 2 phases. The first phase was witnessed on 7 June, with around 405 critical cases, and 91% of infants below 5 years old were dead . FurtherTran Minh Dien, vice director of the National Hospital for infants, announced that 58.7% of cases were occurred by EV71 in Vietnam. In South Korea, the first infected patient with HFMD was recognized in spring 2009 and hospitalized; after some days it was found that all the patients were dead owing to severe complications. Besides these, HFMD occurred in Japan in 1997–2000 and Singapore and Malaysia in the year 1997. Chen et al. demonstrated that EV71 is mostly seen in Asia–Pacific countries. Moreover, the C4 genotype of EV71 is currently found in China, Hong Kong, Korea, and Vietnam, while B5 was found in Japan, Malaysia, and Taiwan.In Australia, in June 2013 around 100 children were infected by EV71, of which four cases were detected in Sydney. In Cambodia, between April and July 2012, the fatality rate was 64, and 2 survived, who were under 7 years old. Infected children died in between 24 h, and symptoms were mainly respiratory problems, fever, and neurological abnormalities. On 6 July 2012, out of 24, 15 were found positive with EV71. After that, the infection rate gradually decreased. On 15 July 2012, WHO declared that Cambodia is free from EV71 .The Enterovirus 71 genotypes are detected in different countries, as shown in Table According to Solomon et al.  genogrouFurther, it was found that the change of C4a to C4b is the major reason for increasing neurovirulence epidemics in China , 42. FroThe most frequently observed disease in EV71-infected patients is pyrexia (eighty-one per cent) with a skin rash (seventy per cent), insomnia (sixty-six per cent), vomiting (sixty-two per cent), lethargy (forty per cent), myoclonus (sixty per cent), and ataxia (forty-two per cent). CSF  evaluations were carried out in 47 clients, where 45 were pleocytosis, 94/ml median, 15–920/ml range among them. However, none of them had viruses isolated from specimens of CSF. The most frequently diagnosed vector of CA16 was HFMD, while CA5, CA9, and CA10 were statically identified as HFMD as a source among other recognized HFMD enteroviruses.The EV71 and CA16 are highly neurotropic viruses that have caused severe CNS issues in patients, and these CNS complications have been identified as the primary causes of fatal HFMD. From the clinical experiments in Western Australia, and Japan, Malaysia indicate that hand–foot–mouth disease (HFMD) rashes may be variable because of CA16 and EV71. Rashes or erythema occurs due to CA16 in the arms and legs, of larger vesicles than the EV71 virus, where the rash is most commonly popular and petechial in those viruses. The infection of three to four days was characterized, as the disorder in the mouth, gums, palate, papulosicles and the distribution of vesicular enanthemum in the labia, buttocks, feet seen in EV71 patients. Some major clinical symptoms are briefly shown in Table The flow chart of the pathogenesis of Enterovirus 71 is presented in Fig. From the study of autopsy in Malaysia, inflammation was found in the spinal cord grey matter, brainstem, hypothalamus, subthalamic and dentate nuclei . NeuroloIt has also been reported that abnormal cytokine activation produces severe inflammation in the lungs . From seSome EV71 infections are not only symptomatic but also asymptomatic and can be controlled by vaccine . Till daOn the other hand, a formalin-inactivated EV71 vaccine is expressed as an alum-adjuvant vaccine which causes cross-neutralizing antibody comebacks in a phase III trial. As per reports, about 30,000 children in China were cured in phase III clinical trials of inactivated EV71 vaccines. In December 2015, China’s Food and Drug Administration officially investigated 2 inactivated EV-A71 vaccines which inhibited serious HFMD in children. On the other hand, formalin-inactivated EV71 vaccines were developed in Taiwan and Singapore which underwent Phase I clinical trials, and the results were excellent for preventing of EV71 infection. Although inactivated EV71 vaccines were mostly used for safeguarding the children, they had the potential to inhibit the replication of the viral genome.The virus-like particles (VLPs) vaccines are completely different from the inactivated whole-virus EV71 vaccines. VLPs vaccine possesses all surface epitopes of the capsid protein and is very less effective than inactivated vaccines . In addiEV71 is a life-threatening disease for infants and children across the world. This viral infection is fast-spreading in Asia, causing chronical health hazards like HFMD, neurological abnormalities among children. HFMD is a viral and transmissible disease. Children get infected in 3 days after exposure to the virus and remain infectious until 10 days. The major symptoms of this infection are fever, runny nose, sneezing, cough, rashes in skin, ulcer in mouth and muscle aches, etc.To date, no specific treatments have been found for EV71 virus. Children with mild symptoms are one of the reasons for non-polio enterovirus infection. Most of the children recover completely, but some get affected with serious complications or lethal to the virus. Therefore, there is an urgency of treatment for these viral infections. EV71 vaccination is a priceless gift for children in Asia–Pacific as well as across the world. Clinical studies on inactivated EV71 vaccines have proven to be a useful weapon in the battle against EV71 infection-associated severe HFMD outbreaks. Companies in Asia lead the development of EV71 vaccines, demonstrating that vaccine R&D and assessment in these Asian nations have improved. Nonetheless, market entrance approvals and applications in other countries with significant targeted populations of babies and children face technological and legislative obstacles. Vaccine availability, the possibility of introducing the EV71 vaccination into EPI, and the harmonization of vaccine standards are the hurdles. As a result, international collaboration is critical for controlling the spread of the EV71 vaccination in underdeveloped countries. WHO should take the lead in developing EV71 vaccine quality control and assessment standards.The good news is that the National Institutes of Health's National Institute of Food and Drug Control and the National Institute of Biological Standards and Control are working together to develop WHO neutralizing antibody standards for EV71 vaccines, which could help standardize vaccine quality control and evaluation. Only the Japanese encephalitis (JE) vaccine developed by the Chengdu Institute in China has received WHO pre-qualification. The world still has a long way to go before the cost-effective EV71 vaccine is widely available.


Induced pluripotent stem cells (iPSCs) have been considered an essential tool in stem cell research due to their potential to develop new therapies and technologies and answer essential questions about mammalian early development. An important step in generating iPSCs is selecting their precursor cell type, influencing the reprogramming efficiency and maintenance in culture. In this study, we aim to characterize bovine mesenchymal cells from adipose tissue (bAdMSCs) and fetal fibroblasts (bFFs) and to compare the reprogramming efficiency of these cells when induced to pluripotency. The cells were characterized by immunostaining , induced differentiation in vitro, proliferation rates, and were subjected to cell reprogramming using the murine OSKM transcription factors. The bFFs presented morphological changes resembling pluripotent cells after reprogramming and culture with different supplementation, and putative iPSCs were characterized by immunostaining . In the present study, we demonstrated that cell line origin and cellular proliferation rate are determining factors for reprogramming cells into pluripotency. The generation of biPSCs is a valuable tool to improve both translational medicine and animal production and to study the different supplements required to maintain the pluripotency of bovine cells in vitro. Stem cells have two main characteristics, the capacity for self-renewal and the potential to differentiate into other cell types, and are widely studied for their use in veterinary and human regenerative medicine ,2. The pInduced pluripotent stem cells (iPSCs) were first reported from murine, followed by human fibroblasts, transduced with a retroviral vector containing the transcription factors OCT4, SOX2, KLF4, and c-MYC (OSKM) ,10. The The generation of iPSCs in the bovine model (biPSCs) can greatly benefit veterinary and human regenerative medicine studies, serving as a biomedical model or bioreactors ,21. It cTo characterize the biPSCs, the detection of usual pluripotent markers, including mainly OCT4, SOX2, and NANOG, and differentiation into embryoid bodies or teratoma assays are usually performed, and the standard gold test is the chimera production ,31. The Herein, two cell types, bovine fetal fibroblasts (bFF) and bovine adipose tissue mesenchymal stem cells (bAdMSC), were characterized and then reprogrammed using OSKM. The efficiency of reprogramming in different culture conditions (LIF or bFGF) was morphologically analyzed. Albeit further molecular analysis and an increased number of animals studied are needed to better support our findings, the results presented herein may contribute to the selection of cell origin, the establishment of a reprogramming protocol, and adequate culture conditions for biPSC generation and maintenance. The generated cells can be further used for the in vivo and in vitro study of diseases and genetic improvement, for example, for the in vitro generation of gametes and embryos.2, and maximal humidity , analyses were performed in biological triplicates, and photo-documentation was performed with a Nikon Eclipse TS100 microscope equipped with a Nikon DS-Ri1 camera and the NIS-Elements F (v 2.1) software.All procedures were approved by the Ethics Committee on the Use of Animals of FZEA/USP (n° 3526250717). Unless otherwise stated, cells were cultured at 38.5 °C, 5% CO3 of adipose tissue [Briefly, bovine fetal fibroblasts (bFF) were isolated from a 50-day gestation fetus , and adie tissue  from an 7 cells/mL and cultured under standard conditions for 2 h, when the differentiation medium was added and prepared according to the manufacturer’s instructions and refreshed every four days for two weeks. After the differentiation process, cells were fixed and stained using Alcian blue dye  for chondrogenesis differentiation detection and were photo-documented.bFFs and AdMSCs were submitted to induced differentiation using the StemPro commercial kit , as previously described  and acco4 cells/well and incubated for two days. Every 48 h, the cells were dissociated, counted using a Neubauer chamber, and reseeded at the initial density (3 × 104 cells/well) [DT) was calculated in hours using the equation [As previously described , for douls/well) . This prequation :DT=(T−T0T − T0): number of hours cells were cultured between passages,  were fixed with 4% paraformaldehyde. The cells were analyzed for typical mesenchymal marker CD90 , and for pluripotency markers: SSEA1 , SSEA3 , and SSEA4 . Briefly, the cells were washed with buffer (0.1% Tween + 1% Bovine Serum Albumin (BSA)). Subsequently, blocking with 10% BSA was performed, the cells were washed with buffer, and each well of the plate was incubated overnight with the specific primary antibody. After the incubation period, the cells were washed with phosphate buffer saline (PBS), incubated for 1 h with a secondary antibody , washed with buffer, and incubated for 5 min with Hoechst . Subsequently, washing with PBS was performed, and the cells were photo-documented as previously described.bFFs and AdMSCs  in iPSCs media composed of KnockOut Dulbecco’s modified Eagle’s medium , supplemented with 20% KnockOut Serum Replacement , 1% L-glutamine (Life Technologies), 1% nonessential amino acids (Life Technologies), 1% antibiotics , and 3.85 μM β-mercaptoethanol (Life Technologies). Media culture was supplemented with 10 μg/mL human bFGF  or 1000 U/mL LIF .After six days, the cells were harvested using Tryple Express (Life Technologies) and 2 × 102.Cells were kept in iPSCs media for approximately 20 days or until colonies were formed, and the media was refreshed every two days. The efficiency of iPSC colony generation was measured by counting the number of colonies formed in relation to the number of cells initially plated to the transduction. After a first manual splitting of iPSC colonies, clonal lineages were enzymatically dissociated, replated, and cryopreserved. Reprogrammed cells were kept in culture for at least 21 passages at 38.5 °C and 5% COFor alkaline phosphatase (AP) detection, the commercial Alkaline Phosphatase Detection Kit  was used. Briefly, colonies were fixed and analyzed according to the manufacturer’s instructions. Colonies stained in pink were considered positive. AP positive clonal lineages were further analyzed for the detection of NANOG, OCT4, and SOX2.biPSCs colonies were washed with PBS, permeabilized with 0.1% Triton X for 20 min, washed with PBS, and blocked with 1% BSA/0.05% Tween for 1 h and labeled for 1 h with primary antibodies: NANOG , OCT4 , and SOX2 . Cells were then washed with 0.05% Tween, incubated for 1 h with a secondary antibody (anti-rabbit Alexa Fluor 488-1:500 dilution), washed as described above, and labeled with Hoechst 33342 (Sigma-Aldrich—1:1000 dilution) for 5 min. After a final wash with PBS, the cells were photo-documented.To evaluate the plasticity of bFFs and bAdMSCs, the cells were induced to chondrogenic lineage. The analysis of cell differentiation was performed based on the morphological comparison between the supplemented cells and the control cells . The celImmunocytochemistry was performed to detect pluripotent and multipotent markers in the bFFs and bAdMSCs. The CD90 marker  was detected in both, but not the SSEA-1, SSEA-3, and SSEA-4 .bFFs presented a shorter doubling time , approxiAfter pluripotency induction, only bFF cells showed morphological signs of reprogramming 10 days after transduction. The reprogrammed cells were cultured in different supplements, bFGF or LIF, and were analyzed regarding their morphology . The repColonies supplemented with LIF showed compacted colonies with well-defined borders and, based on their morphology, were selected for further characterization, such as the detection of alkaline phosphatase (AP) and markers related to the pluripotency: NANOG, OCT4, and SOX2 .Bovine iPSCs have already been reported in several divergent studies regarding reprogramming methods and culture conditions, and the majority were derived from fetal or embryonic bovine fibroblasts ,36. In tThe bFFs and bAdMSCs were positive for CD90, which is typical of mesenchymal stem cells , and theFaster cell proliferation rates are among the main characteristics of stem cells . In thisInterestingly, after inducing the bFFs and bAdMSCs into pluripotency, only biPSCs derived from bFF cells were generated. Thus, besides the reprogramming process being multifactorial dependent, to our knowledge, there are no reports of iPSCs derived from bAdMSCs. Ruiz et al.  reportedAfter pluripotency induction, the bFF cells showed clear signs of cell reprogramming, such as dome-shaped and compact colonies with well-defined borders, similar to those previously reported by Bessi and collaborators  with bFGTo maintain the pluripotency of biPSCs during the in vitro culture, supplementation is necessary. LIF is responsible for inhibiting differentiation and stimulating self-renewal , while bOur study demonstrated that the efficiency in generating biPSCs cells might be influenced by the origin of the somatic cells and possibly the donor age. The biPSCs generated from bFF cells were characterized based on their morphology and immunophenotype. These bovine reprogrammed cells can be used in translational studies to improve animal reproduction and for veterinary or human regenerative medicine.In summary, our results showed that the bFFs and bAdMSCs have the potential to differentiate into chondrocytes after the in vitro induction, were CD90 positive, and the bFFs presented a shorter doubling time. The bAdMSCs, however, showed a higher doubling time rate and no signs of reprogramming. In our conditions, the bFFs seem more promising for in vitro cellular reprogramming, as they presented expected morphological changes such as the formation of well-defined colonies, and the putative iPSCs generated were positive for alkaline phosphatase and markers related to pluripotency . Nonetheless, additional morphological analysis including more animals, and molecular analyses of the data presented here, could further characterize the state of the pluripotency of generated cells and complement the results gathered here. Finally, we conclude that multiple aspects, such as the cell tissue source, the age of the animal, and the proliferation rates can influence the generation of biPSCs. Moreover, our results can contribute to the development of pluripotent cells in large animal models.


Background: The role of platelets in the immune response against influenza has been raised, and a diagnostic or prognostic value of platelet parameter abnormalities, including platelet count (PLT), or mean platelet volume (MPV), has been suggested. The study aimed to analyze the prognostic value of platelet parameters in children hospitalized due to laboratory-confirmed influenza. Methods: We retrospectively verified the platelet parameters , and the clinical course . Results: An abnormal PLT was observed in 84 out of 489 laboratory-confirmed cases . Patients’ age correlated negatively with PLT (rho = −0.46) and positively with MPV/PLT (rho = 0.44), while MPV was not age-dependent. The abnormal PLT correlated with increased odds of complications (OR = 1.67), including LRTI (OR = 1.89). Thrombocytosis was related to increased odds of LRTI (OR = 3.64), and radiologically/ultrasound-confirmed pneumonia (OR = 2.15), mostly in children aged under 1 year . Thrombocytopaenia was related to antibiotic use (OR = 2.41) and longer hospital stays (OR = 3.03). A lowered MPV predicted a tertiary care transfer (AUC = 0.77), while MPV/PLT was the most versatile parameter in predicting LRTI (AUC = 0.7 in <1 yo), pneumonia (AUC = 0.68 in <1 yo), and antibiotic treatment (AUC = 0.66 in 1–2 yo and AUC = 0.6 in 2–5 yo). Conclusions: Platelet parameters, including PLT count abnormalities and MPV/PLT ratio, are related to the increased odds of complications and a more severe disease course, and may add important data in assessing pediatric influenza patients, but should be interpreted cautiously due to age-related specificities.  Streptococcus pneumoniae and Staphylococcus aureus are the most frequent bacteria responsible for suprainfections , while the remaining cases are caused by a wide spectrum of bacterial pathogens [Influenza is one of the most important viral pathogens that may cause a varied disease course; from a self-limiting illness to severe complicated cases, requiring intensive care, or resulting in a fatal outcome . The infathogens . Therefoathogens , althougathogens .The immune response against influenza infection is of particular interest, and the role of platelet activation in the host defense is being investigated. The platelets seem to internalize the influenza virus, and a cross-communication between the platelets and the neutrophils mediates the immune response, including a complement path . HoweverThe platelet count acts differently under specific conditions, depending, inter alia, on the aetiological factor . In inflAnother key parameter is the mean platelet volume, alongside with the mean platelet volume/platelet count ratio (MPV/PLT), as the MPV correlates with platelet activation . High MPResearch to date is scarce and contains a rather low number of pediatric patients, and there is a definite need for improvements in influenza diagnostics. In this study, we sought to analyze the prognostic value of platelet parameters  in a huge group of children hospitalized due to influenza with regards to the prediction of complications , and the clinical course of influenza .The study was granted permission by the local Ethics Committee at the Centre of Postgraduate Medical Education, Warsaw (permission number 91/PB/2020 issued on 15 June 2020). The study was conducted in accordance with the Declaration of Helsinki with its later amendments, and parents’/tutors’/patient’s consent was waived due to its retrospective character. The electronic medical charts of children hospitalized at the Department of Pediatrics (aged 0–18 years old), Bielanski Hospital, Warsaw, between January 2013 and March 2020 were verified. The search aimed to retrieve patients with one of the ICD-10  codes corresponding to influenza: J10 or J11 with extensions. Only children with laboratory-confirmed influenza were eligible for the study; the diagnosis was confirmed with the rapid influenza diagnostic test (RIDT), and/or reverse transcription-polymerase chain reaction (RT-PCR), performed in samples from a nasopharyngeal swab. The RT-PCT was a reference method and was considered conclusive in dubious cases, since both false positive and false negative results of RIDT may occur ,23,24. APatients with a complete blood count were included in the analysis. The blood count analysis was performed with the use of Sysmex XN1000/Sysmex XN550  since 10 April 2014, and before that date, with the use of Sysmex XT2000i . CRP was measured with the Cobas 6000 analyzer  with a limit of detection of 0.1 mg/L, while procalcitonin was measured with the Cobas e411 analyzer  until 4 August 2016, and with the Cobas 6000 analyzer  from 5 August 2016; the limit of detection in both cases was 0.02 ng/mL. Capillary blood gas analysis was performed with the use of the Roche Cobas b121 and b221 analyzer  until 26 January 2016, and with the RAPIDLab 348EX Blood Gas System  from 27 January 2016. All the analytic processes were conducted according to the manufacturers’ instructions.The exclusion criteria consisted of: an immune deficiency , immunosuppression , a history of neoplasm, a hemodynamically significant heart disease, and a discharge on patient’s/parent’s/tutor’s demand. For the purposes of analyzing the presence of pneumonia, the lack of a radiographic confirmation of pneumonia was considered an exclusion criterion. 3, while thrombocytopaenia was <150,000/mm3 [Baseline characteristic assessments included the age, gender, duration of symptoms prior to hospitalization, and, separately, duration of fever prior to hospitalization. The following platelet parameters were analyzed: platelet count (PLT), mean platelet volume (MPV), MPV to PLT ratio, and PLT to lymphocyte (PLT/LYM) ratio. In line with the previously published studies in this field, thrombocytosis was defined as a platelet count > 450,000/mm,000/mm3 .Both age and influenza severity might be expected to influence platelet parameters, thus the analyzes were performed in the whole study group, and patients were furtherly divided into age subgroups. Firstly, due to practical implications, influenza risk groups were taken into consideration: children under 5 years old, and especially those younger than 2 years old are at higher risk of a more severe disease course, and corresponding age groups were created . MoreovePatients were analyzed in terms of the presence of complications: AOM, LRTI, and radiologically-confirmed pneumonia. All the endpoints, i.e., AOM, LRTI, and pneumonia were defined according to the official Polish guidelines :Acute otitis media was defined as the presence of acute signs and/or symptoms of acute otitis media  and an otoscopy result: the presence of fluid in the tympanic cavity  and a reddening of the tympanic membrane. LRTI was defined as a final diagnosis of bronchitis, bronchiolitis, or pneumonia. Bronchitis is recognized when cough (productive or unproductive) is accompanied by wheezing or rales on auscultation, while bronchiolitis is defined as the first episode of the restriction of bronchioles . Pneumonia was defined as the presence of clinical signs and/or symptoms . Tachypnea was defined as >60 breaths/minute in neonates (0–1 months), >50 breaths/minute in children 1–12 months, >40 breaths/minute in those aged 12–59 months, and >25 breaths/minute in children aged 60 months or older. For the purposes of the pneumonia analysis, we included only patients with a confirmation in a chest X-ray or lung ultrasound, meaning the presence of any of the following: consolidation, linear densities, patchy densities, parenchymal infiltrates, or pleural effusion or (in the case of the lung ultrasound), the presence of hypoechogenic lesions, pleural line abnormalities, bronchogram sign, or an impaired lung respiratory mobility (absence or decrease in “lung sliding”).A second group of endpoints consisted of the clinical course assessment, i.e., length of stay (LOS), need for antibiotic treatment, need for intensive care unit (ICU), or other tertiary care reference center transfer related to the disease severity, and an unfavorable outcome. The LOS values were dichotomized upon the median value in order to be presented in a binary mode.The analysis of the confounding factors included gender and hydration status. Hydration status was assessed by pH, bicarbonate, and base excess levels from capillary blood gas measurements, which was proved to reflect arterial blood gas value strictly ,31. t-test or the Mann-Whitney U test) was applied. The Kolmogorov-Smirnov test was used to assess data distribution, and a mean with a standard deviation (SD) or a median with an interquartile range (IQR) was used to present the results according to the data distribution. A corresponding parametric or non-parametric test (the Student The correlation between continuous data was presented as Spearmann’s rank correlation coefficient (rho). The receiver operating curve (ROC) analysis was used to calculate the area under the curve (AUC) with a 95% confidence interval (95%CI). Due to the age-related specificities, the ROC analysis was performed in the whole study group, and then separately in each age group in order to decrease the age influence and to calculate the most accurate cut-off values that could be used in clinical practice . An optimal cut-off value for the prediction of the endpoints was estimated with the Youden index. In the case of statistically significant AUCs under 0.5 , the equation was reversed in order to present easily comparable results , with an emphasis on the destimulating role, marked by the word “lowered” preceding the parameter. A multivariate regression model for dichotomous data was run to calculate the odds ratio (OR) with a 95% confidence interval. p-value under 0.05 was considered statistically significant. The statistical analysis was conducted with the use of Statistica 13.1 software .A During the analyzed period (87 consecutive months) there were 12,639, and 489 children were diagnosed with laboratory-confirmed influenza, corresponding to 3.9% of the total number of hospitalizations. A flowchart of the patients is shown in p = 0.038 and p = 0.033, respectively), with no differences in the platelet parameters . A direct comparison between influenza A and B also showed a slightly shorter duration of symptoms, and a fever in those with type A . The AOM was diagnosed in 88 cases (18%), and LRTI was diagnosed in 154 cases (31.5%), including 128 pneumonia and 26 other than pneumonia cases; 35 patients presented both types of complications, i.e., LRTI and AOM. Eighteen children were diagnosed with pneumonia on a clinical basis only, i.e., without a radiological or ultrasound confirmation, thus, they were excluded from the analysis on pneumonia. Finally, radiologically confirmed pneumonia was diagnosed in 110 out of 471 children (23.4%). The highest frequency of complications was observed in mixed influenza A and B infection , followed by type A , and type B . Antibiotics were implemented in 186 out of 489 children (38%), with the highest use in children coinfected with influenza A and B , followed by type A , and type B . Four patients required a transfer to a tertiary care unit . The outcome was favorable in every patient. 3/µL (IQR: 190–320), and an abnormal platelet count was observed in 84 children (17.2%), including 44 cases (9% of the whole study group) of thrombocytopaenia and 40 cases (8.2%) of thrombocytosis (p = 0.015), although the duration of fever prior to hospitalization did not differ between the groups, neither did the length of stay. Thrombocytopaenia was observed in 11.5% of influenza B cases, 8.4% influenza type A, and 6.7% mixed influenza A and B. Thrombocytosis was seen mainly in influenza type A (9.2%), followed by A and B (6.7%), and type B (4.8%) infection (The median platelet count was 244 × 10ocytosis . Childrenfection .The median MPV reached 9.7 fL (IQR: 9.2–10.3) in the whole study group, while the median MPV/PLT was 0.0403 (IQR: 0.0295–0.0527), and the median PLT/LYM ratio equaled 98.88 (IQR: 63.9–166). There was a positive correlation between the patients’ age and the MPV/PLT (rho = 0.44) and the PLT/LYM (rho = 0.27), and a negative correlation with the PLT (rho = −0.46), while the MPV did not correlate with the age . An intep = 0.036), but thrombocytopaenia nor thrombocytosis alone did not significantly increase the odds (p = 0.01), and thrombocytosis in specific  were significant with regards to LRTI. Thrombocytosis  was related to an increased risk of radiologically/ultrasound-confirmed pneumonia . Platelet count abnormalities did not correlate with the presence of AOM. On the other hand, for the prediction of antibiotic use, only a lowered platelet count was significant , while the thrombocytosis and the abnormal platelet count remained insignificant. Similarly, children with thrombocytopaenia were at higher risk of a longer (than median) hospital stay . None of the platelet count abnormalities correlated with the tertiary care center transfer.In a multivariate regression model, the abnormal platelet count was correlated with increased odds of complications , but the odds were insignificant in the remaining age groups. Similarly, LRTI odds were increased in children under 1 year of age , but the odds in the other age groups remained insignificant. Furtherly, a regression model was run in the aforementioned age groups. Thrombocytosis was related to an increased odds ratio of pneumonia only in children under 1 year of age , had a longer history of signs and/or symptoms , and fever , required longer LOS , and presented with lower MPV , a lower MPV/PLT ratio , a lower PLT/LYM , while the total platelet count did not differ between the groups (p = 0.075).Children with complications were younger , followed by lowered MPV , and lowered MPV/PLT , followed by a lowered MPV , and PLT count . The cut-off values estimated with the Youden index exhibited the following sensitivity/specificity: 38 and 92% for MPV/PLT (at the cut-off of 0.02), 73% and 50% for MPV (at the cut-off of 9.9 fL), and 32 and 89% for PLT , respectively, whereas the age did not differ. Among the platelet parameters, only the PLT/LYM ratio was significantly lower , and adequately, a significant AUC in the ROC curve analysis was proved only for a lowered PLT/LYM , while the other parameters remained insignificant. Children with AOM had signs/symptoms of fever for a longer period of time , had a longer history of signs/symptoms and fever , and required longer hospital treatment . They had a higher median platelet count , lower MPV/PLT , and lower PLT/LYM , while MPV did not differ between the groups.Children with LRTI were younger  for PLT count, and lowered MPV/PLT , and 0.57  for lowered PLT/LYM, while in the case of MPV, it remained insignificant. Similar to the prediction of complications, the age group analysis showed a significant AUC only in those under 12 months of age, and it equaled 0.7 for lowered MPV/PLT , and 0.67 for the PLT count , while it was insignificant for the other two platelet parameters. The cut-off values showed a sensitivity and specificity of 44% and 93%, respectively; at the cut-off point of 0.02 for MPV/PLT, and 58% and 72% at PLT = 371 thousand/µL. The AUC for predicting LRTI equaled 0.59 , had longer LOS , but showed only lower MPV/PLT , and a corresponding analysis did not reveal any statistical significance in terms of predicting pneumonia. According to the age groups, a significant relationship was seen for lowered MPV/PLT  in children under 12 months of age , whereas none of the parameters were significant in the other age groups. Patients with a radiological/ultrasound confirmation of pneumonia presented signs/symptoms and fever longer , longer history of signs/symptoms , fever , and a longer hospital stay . Children with antibiotic treatment had a higher median platelet count , lower MPV , and lower MPV/PLT , whereas the PLT/LYM was insignificant.The need for antibiotic therapy was related to a younger age , a lowered MPV , and a lowered MPV/PLT  showed a significant AUC in predicting the need for antibiotic treatment, while the PLT/LYM was insignificant. An age group analysis revealed a statistical significance for PLT, MPV, and MPV/PLT in those aged 1–2 years , and for PLT and MPV/PLT in children aged 2–5 years . The PLT cut-off values of 284 thousand/uL and 231 thousand/uL in children aged 1–2 years and 2–5 years indicated a sensitivity/specificity of 64%/69% and 61%/58%, respectively. The MPV/PLT at the cut-offs of 0.03 and 0.04 showed 44%/81% of sensitivity/specificity and 51%/60%, respectively. In children aged 1–2, the MPV at the value of 9.5 fL had 67% and 63% of sensitivity and specificity, respectively. The PLT count , while there were no differences between their platelet parameters. The ROC curve analysis showed a significant AUC for the MPV , while the other parameters were insignificant. At the cut-off value of 9.9 fL, the sensitivity was 100%, with a specificity of 56%  and thrombocytosis is in line with the higher platelet counts previously reported by Lee in 72 children with A/H1N1 pandemic influenza complicated by pneumonia, compared to those without pneumonia . We obseApart from the LRTI, another potential use of the platelet count may be a differentiation between bacterial and viral infections. While patients with antibiotics had a higher median platelet count, thrombocytopaenia, as a sole predefined platelet count abnormality, was related to increased odds of antibiotic use. A study on a group of adults hospitalized because of AH1N1 and CAP revealed higher platelet counts in those with a bacterial coinfection , but HsiA complex relationship between the platelet count and the clinical course is reflected in our study by the odds of complications; while an abnormal platelet count was correlated with an increased odds ratio, none of the abnormalities alone  were significant to predict the presence of complications. Another parameter analyzed, the MPV, acts differently with regards to the various endpoints; a decreased MPV, correlated with the presence of complications and antibiotic therapy, while an increased MPV was prognostic of the need for a tertiary reference center transfer. Of interest, the relationship between antibiotic use and the MPV was seen only in children aged 12–23 months, but it demonstrated the broadest AUC compared to other platelet parameters. Limited data regarding the MPV and respiratory complications are available; previous studies on pneumonia in adult patients showed that a high MPV , or its Among the platelet parameters studied in this research, the MPV/PLT ratio seems to be the most versatile, in general, and to present the highest prognostic value in particular cases. Moreover, due to the inclusion of two parameters in the ratio , the influence of age-related fluctuations on any of them is decreased. In our series of patients, the MPV/PLT ratio showed the ability to predict complications, and the need for antibacterial therapy, and had the most robust area under the curve for predicting complications (with LRTI as a specific condition), as well as antibiotic treatment. Our results showing a lowered MPV/PLT seem contradictory to the studies by Sayed and Golwala, who found a relationship between an increased MPV/PLT and higher mortality  ,45, whilA similar diagnostic value of an increased MPV/PLT ratio was revealed in the studies by Han and Fei, who compared children diagnosed with infectious mononucleosis or influenza, respectively, against healthy controls ,46. HoweSurprisingly, the MPV/PLT was the only platelet parameter that correlated in the ROC analysis with the presence of radiologically-confirmed pneumonia, and this was seen only in a subgroup of children under 12 months of age, whereas it remained insignificant in the other age groups and the whole study group. The need for antibiotic therapy correlated also with a decrease in MPV/PLT ratio, which is contradictory to the study by Lee, who reported higher MPV/PLT in patients with increased procalcitonin levels , that arThe fourth platelet parameter assessed in previous studies is based on a relationship between the PLT and lymphocytes. Fei and colleagues analyzed platelet parameters in terms of influenza type A (not B), including 191 children with laboratory-confirmed influenza A, compared their laboratory characteristics with children without influenza A who had or had not influenza-like illness (ILI) symptoms, and observed the highest diagnostic value (the largest area under the curve) for lowered LYM*PLT in distinguishing influenza A from non-influenza A ILI. It might probably be a disease-specific condition since influenza is related to the decrease in both lymphocytes and platelet count . A relat3 and thrombocytosis > 450,000/mm3) was seen in 24.3% of patients with influenza type B, and 11.4% with influenza A, while in our study, the frequency of an abnormal platelet count was similar: 17.6% in patients with influenza A, and 16.4% in children with type B, and, unexpectedly, in a lower percentage of children with mixed A and B coinfection (13.3%). A generally higher frequency of thrombocytopaenia rather than thrombocytosis reported in a study by Kim was not confirmed in our group of patients; we observed only a slight predominance of a lowered platelet count (9% vs. 8.2%) [There are several factors influencing platelet parameters. Kim et al. conducted huge research on platelet abnormalities during respiratory viral infections, which enrolled, inter alia, 4618 patients aged 0–9 years. An abnormal platelet count  . Nor dids. 8.2%) . Corresps. 8.2%) . The frequency of platelet abnormalities may depend on the study settings, host and virus characteristics, named not only by the influenza types but also by the subtypes/lineages. A study by Yan, for example, revealed that A/H3N2 patients had lower PLT counts than other influenza patients (a study conducted in patients aged 0.5–95 years) . Our invInterestingly, in the study by Kim, the highest prevalence of thrombocytosis was observed in infants (26.1%), then dropping rapidly, and children over 2 years of age presented an increasing tendency towards thrombocytopaenia . CorrespIn our study, we also analyzed the relationship between the patient’s age and the PLT number and found a moderate negative correlation (rho = −0.46). The recognition of age-related specificities is mirrored in the statistical significance of AUCs in the age groups. In the majority of cases, in children under 12 months of age this relationship was strong enough to result in statistical significance for the whole group, and, consequently, AUCs showed the highest values in this particular age group. An analysis of platelet parameters should be thus considered mostly in the youngest group of patients. n = 6381 in the analysis by Muthuri) [The group of patients included in our research presented with complications in more than 42% of cases, which seems to be similar to other groups of hospitalized children with influenza. The frequency of radiologically-confirmed pneumonia in our study (over 23%) is in line with the 2009 pandemic analysis, which revealed pneumonia in over 20% of pediatric patients with A/H1N1pdm09 , and is Muthuri) , reflectThere are several important strengths and limitations to this study. The research succeeded to gather a huge pediatric group of patients hospitalized due to laboratory-confirmed influenza, which, in our belief, may be considered representative. However, it is only a single-center study, and further multicenter studies are required. Secondly, its retrospective character did not allow the PLT and/or MPV to influence the clinical decision-making process, on the other hand, the clinical course was not influenced by the study, especially in terms of antibiotic therapy. Thirdly, we did not assess platelet distribution width (PDW), which might add more information, since it is changing especially under pathological conditions . We did In conclusion, an abnormal platelet count was related to the increased odds of complications (including LRTI), thrombocytopaenia was related to the increased risk of longer LOS and antibiotic use, while thrombocytosis with the risk of LRTI and radiologically-confirmed pneumonia. The MPV/PLT ratio had the most robust AUC for predicting complications (with LRTI as a specific condition), and antibiotic treatment, while MPV correlated with tertiary reference center transfer. However, the calculated AUCs showed at the very best rather moderate usefulness of every single parameter, and a practical indication of cut-off values is complicated due to, inter alia, age-related differences. To conclude, platelet abnormalities in the course of influenza may contribute important data in the assessment of pediatric influenza patients, and further, prospective multi-center studies are necessary. A broad potential research value might be expected in the field of bacterial coinfections, and an in-depth analysis of the pathomechanism underlying platelet action is required.