The aim and design of the study
The study was carried out among 134 women giving birth in hospitals in Lublin, Poland. The women were residents of Lublin Province, which is located in the east of Poland, near the country’s border with Belarus and Ukraine. The eastern border of the region forms part of the eastern border of the European Union. The key climate issue in the region are emissions from individual home heating systems and road transport. Elevated levels of particulate matter (PM10 and PM2.5) in the air are mainly recorded in cold months, when pollution levels from low-emission sources are higher. The environmental levels of the heavy metals monitored in the region are relatively low, below the target and limit values. Lublin Province ranks in the middle of Polish regions in terms of its pollution levels18,19. The study was carried out at the John of God Independent Public Regional Hospital in Lublin and the Cardinal Stefan Wyszynski Regional Specialized Hospital in Lublin. Research was suspended from November to December 2020 due to introduction of lockdown in Poland, related to the emergence of SARS-CoV-2, for this reason it is difficult to calculate the minimum number of people in the sample.
The umbilical cord blood samples analyzed in the study were collected between 2020 and 2021. The study was carried out once consent had been given by the heads of the delivery rooms where the study was conducted.
All women who consented to participate in the study and met the inclusion criteria took part in the study. The criteria for inclusion in the study were as follows: place of residence—Lublin County, age 18 or over, single, uncomplicated pregnancy, delivery at ≥ 38 gestational weeks, patient consent. The exclusion criteria were as follows: tobacco use, use of psychoactive substances, diagnosis of cancer or other disease complicating pregnancy, long-term occupational exposure to heavy metals, multiple pregnancy, maternal age under 18 years. A total of 200 women in labor were initially recruited for the study. However, as some of the women did not meet all the inclusion criteria, 134 blood samples were ultimately analyzed in the study.
The anthropometric data of mothers and newborns included in the study was collected by means of a diagnostic survey using a standardized questionnaire. The following data relating to the women studied was collected: age, body weight before pregnancy and pregnancy weight gain. The information collected relating to the newborns included the following anthropometric parameters: birth weight (in grams), birth length (in centimeters), head circumference and chest circumference at birth (in centimeters).
The head circumference is measured with a centimeter tape in the fronto-occipital dimension at the level of the frontal tuberosity and the occipital protuberance. The head circumference of a eutrophic newborn is 34–35 cm. The measurement of the chest circumference is carried out on the front wall of the chest at the level of the nipples, on the back at the line of the lower angles of the shoulder blades. The chest circumference of a eutrophic newborn is 32–33 cm.
Measurement of the newborn’s body length begins with the measurement of the SI parietal-neck length—from the posterior (small) parietal along the spine to the beginning of the gluteal sulcus. Then the centimeter should be carried along the lower limb to the knee flexion and further to the newborn’s heel. The length of the body is between 46 and 54 cm.
All the data relating to the women included in the study was provided by them in the questionnaire. Anthropometric measurements of newborns were made by the researchers themselves in the first hours of the newborn’s life (the same measurement results were written in the medical documentation of newborns: Newborn’s health history and Baby’s health book).
Blood was collected from the umbilical vessels, after the umbilical cord had been cut, into metal-free tubes with ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate. Each sample was labelled with the patient’s code number, as specified in the questionnaire and study participation consent form. The samples were then stored at − 25 °C until chemical analysis was performed. Digestion of blood sample was carried out using DigiPREP (SCP Science) heating block. After removal from the refrigerator, samples were left until they reached paper temperature. Then 2 mL of blood was drawn into DigiTUBE, 1.8 mL of 65% HNO3 Suprapur and 3.4 mL deionized water were added. Digestion was carried out in closed vessels at temperature 120 °C for 90 min. Digested samples were filtered and quantitatively transferred to Digitube vessels and filled up with deionized water to 10 mL.
The levels of heavy metals in the umbilical cord blood samples were measured by high-resolution inductively coupled plasma optical emission spectroscopy (ICP-OES) (PlasmaQuant PQ 9000 Elite, Analytik Jena). The correctness of the method was determined based on certified matrix reference materials. Gas flow were set as follows: nebulizer gas 0.6 L/min, auxiliary gas 0.5 L/min, plasma gas 14.0 L/min. The following analytical lines were used for analysis: Se—196,0280 nm, Zn, 206,2000 nm, Cu 213,5981 nm, Pb 220,3534 nm, Cd 228,8018 nm, Mn 257,6100 nm, Cr 267,7160 nm and the signal reading time was 3 s. Axial reading mode used for all elements, with the exception of manganese, which was determined using attenuated axial mode. The sample transport rate was set at 1.0 mL/min. Concentration measurements for each sample were made in triplicate. The accuracy of the method was determined based on certified reference materials (Seronom Whole Blood L2&L3). The values of the limits of quantification for the analyzed elements in the samples after digestion were as follows: Zn, Se, Pb, Cu—5 μg/L, Cr, Mn—1 μg/L.
The following metals were initially selected for analysis: lead, cadmium, chromium, nickel, zinc, copper, molybdenum, manganese, cobalt, antimony, thallium, vanadium and selenium. The following metals were found in all the umbilical cord blood samples collected: lead, zinc, copper, manganese and selenium. The levels of other metals (cadmium, nickel, molybdenum, cobalt, antimony, thallium and vanadium) were below the limit of detection (LOD).
The precision of the measurements of heavy metal levels in umbilical cord blood was evaluated by the percentage coefficient of variance (CV%). The lowest level of precision was found for the measurements of lead concentration, whereas the highest level of precision was found for zinc and manganese, which was due to the levels of the metals in umbilical cord blood. The results of the measurements for 95% confidence intervals (95% CI) are shown in Fig. 1.
The study was carried out in accordance with the principles of the Declaration of Helsinki and the study design was approved by the Bioethics Committee of the Medical University of Lublin (KE-0254/280/2018).
The study results were collected and statistically analyzed using IBM SPSS Statistics software (PS IMAGO). The values of measurable parameters analyzed in the study were presented using the mean (M), standard deviation (SD), median (me), minimum (Min) and maximum (Max) values. The Pearson correlation coefficient was used to test for associations between selected variables. Regression analysis was conducted to identify predictors of antropometric parameters of studied women and newborns. Regression analysis with moderating factors was performed to evaluate the variables (F-test). The level of statistical significance was set at p < 0.05.
Ethics approval and consent to participate
The study was carried out in accordance with the principles of the Declaration of Helsinki and the study design was approved by the Bioethics Committee of the Medical University of Lublin (KE-0254/280/2018). Informed consent was obtained from all study participants. All methods will be carried out in accordance with relevant guidelines and regulations.