A descriptive, observational study with prospective acquisition of cases was carried out over a period of one year at the Department of Neonatology of the Pereira Rossell Hospital Center (CHPR), in Montevideo, Uruguay. The CHPR obstetric/perinatal department is the most important in the country, assisting around 6000 births per year from the low-income population (6279 births were registered in 2018. Data provided by the Perinatal Computer System, Uruguay). The project was carried out in the following stages: a) Pilot Study: 15 patients were included during a week-period, this phase of the study allowed the verification of all processes, procedure adjustments and training of the team. b) Execution: it involved the inclusion of study samples with measurement of umbilical cord ferritin at birth, the application of the nutritional survey to mothers and the posterior data analysis.
The inclusion of patients was performed according to a checklist system designed to verify the absence of exclusion criteria. during this process and verified the strict match of the inclusion criteria before and after labor. The study was approved by the CHPR’s ethics committee. All patients gave their written informed consent.
The study included term and healthy newborns, with a gestational age equal to or greater than 37 weeks’, from families established addresse in the metropolitan area of Montevideo, who were born in CHPR’s obstetrics/perinatal department during the one-year study period. The exclusion criteria was: newborns born before 37 weeks gestation, severe small for gestational age (less than Percentile 3 of the Fenton charts), major congenital malformations, neonatal depression at birth, risk of specific/nonspecific connatal infection, multiple pregnancy infants, children born to mothers who received iron intravenously, mothers with problematic consumption of psychoactive substances for recreational use, mothers with insulin-requiring diabetes and those newborns who had an early clamping of the umbilical cord, before the first minute of life.
A total of 220 newborns fulfilled the inclusion criterio, 14 did not give their informed consent therefore were directly excluded. In relation to umbilical cord ferritin samples: 2 were not processed, 4 were considered as outliers since results went outside from the lower or upper limits described in the literature. The nutritional survey was applied to 206 mothers, of whom 12 were discarded, considering that the answers offered unreal data in relation to the quantitative variables. Finally, a bivariate analysis of 188 patients with the corresponding variables was possible (Figure 1.)
Regarding the hemogram samples: 23 were not processed due to the inadequate state of the sample and 167 were processed.
Because there are no studies to date investigating the association between beef consumption during pregnancy and ferritin levels at birth, we cannot report information on the proportion of maternal exposure to consumption of beef in the cohort or the OR value to calculate the sample size. In the present study, we use an empirical sample size.
Through a face-to-face interview with the patients, data was collected on the demographic characteristics. Information on the maternal baseline health history (reproductive history, pathological history, lifestyle, exposure to toxic substances, complications during pregnancy, and others) aa well as neonatal relevant data were obtained from medical records. In addition, a family socioeconomic assessment was carried out through a direct interview with the newborns’ parents.
Succeeding a strict cord clamping following one minute of life, umbilical cord blood was drawn, and hemogram and ferritin tests were performed. All samples were collected by trained health personnel. The umbilical cord blood was collected in a heparinized tube to perform the hemogram and a dry tube for the measurement of ferritin. These samples were transferred to the CHPR’s on-call laboratory, where they were stored for a period of less than 24 hours, refrigerated at 4–8ºC, until they were processed. Hemograms were processed in DXH800 hematological quantifier and ferritin was measured by the Cobas 6000 automated equipment, which uses the chemoimmunofluorescence method. All samples were processed following the same protocol. Samples with result values out of the minimun or maximun described values, were processed a second time with the same laboratorial techniques and the results were the same, so they were excluded as outlier data.
A maternal nutritional survey was applied, using a qualitative-quantitative form of frequency of consumption of iron source foods and an approximation of the portions consumed during the last 3 months of pregnancy. The first part of the form collected quantitative information about heme iron sources from animal origin (beef, organ meats, chicken, fish, and pork), non-heme iron sources from vegetable origin (chard and spinach, legumes) and iron fortified foods (products made from iron-fortified wheat flour and iron-fortified dairy products). For a most reliable data collection collection of data, a photographic atlas Visual atlas Guide of food portions and weights (ILSI Argentina, 2018) was used to estimate the weights and volumes of food, preparations and beverages. Mothers’ consumption of pharmacological iron supplement was also explored qualitatively in two groups with good and poor adherence. The total grams of food consumed daily were obtained from the frequency of consumption form and from these the total iron of the diet was calculated, determining both the heme and non-heme fraction. The focus of the results is highlighted in the analysis of beef consumption, since this is the highest consumed in the uruguayan population, consolidating itself as the main source of heme iron. The responses to this survey depended on the mothers’ recall ability and estimation of proportions.Twelve extraordinary quantitative responses were identified, so the interview with the mothers was therefore repeated and given that the responses on the second opportunity did not vary, it was decided to exclude those responses from the analysis.
It was started by controlling the quality of the data collected, it was analyzed through an exploratory analysis of its consistency, outliers were identified, they were confirmed or were excluded when performing a second analysis of the data sources. For continuous variables, normality was tested graphically and using the Shapiro-Wilk test. When the hypothesis of normality was rejected, the data were presented with the median, interquartile range (IIQ: 25–75)), and range with minimums and maximums (Mn-Mx). Qualitative variables were presented as absolute frequency and percentages. The association between the considered exposure factors and the ferritin levels lower or higher than 100ng / ml was tested by means of a bivariate analysis. The Chi-square independence test or Fisher’s exact test for the categorical variables and the Test were used. Wilcoxon-Mann-Whitney waws used for continuous variables. Risk was estimated using OR and their respective 95% CI confidence intervals. A significance level of 5% was considered. The adjustment of a logistic regression model that considers the variables that in the bivariate analysis had a p value <0.20 that allows explaining and predicting cord ferritin levels below or above 100 ng/ml was explored.
Statistical analysis was performed with the R program (R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.)