Preterm labor and related conditions are associated with systemic inflammatory process in the fetus or neonate [
Brain development following an injury is neither static nor a direct consequence of a single event; it actually associates to innumerous cellular and molecular cascades [
This was a prospective observational study of preterm neonates (PTN) from 28 to 32 incomplete weeks of gestational age, who were born from June to December 2014 in a philanthropic hospital in Minas Gerais/Brazil. Infants who were admitted to the neonatal intensive care unit (NICU) and whose parents signed the free and informed consent were enrolled.
Exclusion criteria were (i) 5‐minute Apgar score below 7; (ii) diagnosis of congenital malformations, syndromes, and/or associated diseases; (iii) presence of an acute disorder, including sepsis or necrotizing enterocolitis, at any of the time points; and (iv) death within the first three weeks of life.
Gestational age and birth weight, gender, Apgar scores, infant’s diagnosis at admission in the NICU, conditions associated with the premature birth, and antenatal exposure to glucocorticoids were collected from hospital data.
The study was approved by the Ethics Committee of both the Federal University of Minas Gerais and the Sofia Feldman Hospital. The study protocol did not interfere with medical recommendations or the treatment of preterm neonates in the NICU.
All study participants had biological samples collected at the following time points: at birth (cord blood), 48 and 72 hours of life, and at 3 weeks after birth. Motor development of the neonates was evaluated by means of the Test of Infant Motor Performance (TIMP) that was performed when the babies reached 34 weeks of gestational age.
Umbilical cord blood (5 mL) was collected in sodium heparin tubes (T0). All other samples were obtained simultaneously with other routine laboratory tests in the NICU, without the need for extravenous punctures. At 48 hours (T1), 72 hours (T2), and 3 weeks after birth (T3), venous blood (1 mL) was collected in tubes containing sodium heparin. All samples were immediately centrifuged (5000 rpm, 10 minutes, room temperature), and plasma aliquots were stored at −80°C until assayed.
Urine samples were obtained at the same time points of peripheral blood collection after birth by using a newborn urinary collector, at 48 and 72 hours (T1 and T2, respectively) and at 3 weeks after birth (T3). Urine samples were transferred to 15 mL plastic tubes and immediately centrifuged (3800 rpm, 5 minutes, room temperature). The supernatant was collected and stored at −80°C until analysis.
Measurements of cytokines and chemokines in plasma and urine were performed by cytometric bead array (CBA), according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, USA), using kits for quantitation of inflammatory proteins [
Neurotrophic factors [brain‐derived neurotrophic factor (BDNF) and glial cell‐derived neurotrophic factor (GDNF)] levels were determined by enzyme immunoassay (ELISA), following the manufacturer’s recommendations (R&D Systems, MN, USA). Briefly, monoclonal antibodies, specific for each neurotrophic factor, were incubated in 96‐well plates for 12–18 hours at 4°C. The plates were then washed 3 times with 300
All samples were assayed in duplicate in a single assay to avoid interassay variation. The CBA kits used for the simultaneous quantification of cytokines and chemokines have intra‐assay variations between 4 and 13% for IL‐6, IL‐1
The Test of Infant Motor Performance (TIMP) is a functional motor behavior test used in infants that analyzes child posture and motion. TIMP can be used once the newborn reaches 32 weeks of gestational age and up to four months of corrected age [
In the current study, TIMP was applied when participants reached at least 34 weeks of postmenstrual age. The raw score consisted of the sum of the points obtained in each scale item. Raw scores were converted into
Statistical analysis was performed by the statistical software SPSS version 20.0 (IBM, 2012) and Medcalc version 12.2.1.0 (MedCalc Software, 2012). Continuous variables were described using measures of central tendency and dispersion, and qualitative variables were expressed as absolute frequencies and percentages. Normality was verified using the Shapiro Wilk test. For the time point analysis, the Friedman test was chosen, and for variables with values
Correlation analysis between plasma and urine values, intra‐class correlation coefficient (ICC), Spearman correlation, and Bland Altman plot were used, and receiver operating characteristic (ROC) curves for the two samples were adjusted, in order to choose between the plasma and urine markers to be used in association with the motor development.
Groups with different motor development were compared with
To verify possible confounding factors, the analyses were done with all participants and with the exclusion of three neonates who were not previously exposed to corticosteroids.
This study enrolled 40 infants, 18 (45%) females and 22 (55%) males. Participants were born from 28 to 32 incomplete weeks of gestational age. Prenatal and birth conditions are shown in Table
Mother and infant characteristics.
Variables | Subjects ( |
---|---|
|
|
Preeclampsia | 32 (80.0) |
Other causes | 8 (20.0) |
Predelivery medication | |
Magnesium sulfate + glucocorticoid | 20 (50.0) |
Glucocorticoid | 17 (42.5) |
None | 3 (7.5) |
|
|
Gestational age (weeks)# | 30 ± 1 |
Sex | |
Female | 18 (45.0) |
Male | 22 (55.0) |
Birth weight (grams)# | 1.477 ± 428 |
Apgar scores# | |
1-minute Apgar score | 7 ± 1 |
5-minute Apgar score | 9 ± 1 |
Respiratory distress | 24 (60.0) |
Exposed to antenatal glucocorticoids | 37 (92.5) |
#Values expressed as mean and standard deviation for continuous variables. Number of individuals and percentages for categorical variables.
Newborns presented a significantly decrease in plasma levels of IL‐6, IL‐10, CXCL8/IL‐8, and CXCL10/IP‐10 during the first three weeks of life. The opposite occurred with TNF, CCL2/MCP‐1, CCL5/RANTES, and BDNF. There was a more pronounced increase of TNF and BDNF 48 h after birth (T1). Regarding IL‐12p70, IL‐1
Plasma levels of TNF (a), IL-6 (b), IL-10 (c), and BDNF (d), in preterm infants (
Plasma levels of CXCL8/IL-8, CXCL10/IP-10, CCL5/RANTES, and CCL2/MCP-1 in preterm infants (
In urine samples, IL‐10, IL‐1
Urinary levels of IL-10, IL1-B, CCL5/RANTES, CXCL9/MIG, CCL5/RANTES, BDNF and GDNF in preterm infants (
To determine the relationship between the results of the measurements in plasma and urine samples, ROC curve was used. Either for plasma or urine, ROC curve results showed that no biomarker had an area under the curve (AUC) of 0.9 (Table
Area under the curve (AUC) values for the measurement of inflammatory markers and neurotrophic factors in plasma and urine samples.
Variables | Plasma | Urine | ||
---|---|---|---|---|
AUC values | 95% confidence interval | AUC values | 95% confidence interval | |
TNF | 0.534 | 0.327; 0.741 | 0.620 | 0.438; 0.802 |
IL-12p70 | 0.378 | 0.195; 0.561 | 0.351 | 0.179; 0.523 |
IL-1 |
0.561 | 0.373; 0.749 | 0.853 | 0.722; 0.984 |
CXCL10/IP-10 | 0.443 | 0.246; 0.641 | 0.520 | 0.322; 0.718 |
CCL2/MCP-1 | 0.353 | 0.168; 0.538 | 0.603 | 0.415; 0.791 |
CXCL9/ MIG | 0.458 | 0.270; 0.645 | 0.559 | 0.364; 0.753 |
CCL5/RANTES | 0.278 | 0.115; 0.442 | 0.568 | 0.388; 0.748 |
CXCL8/IL-8 | 0.556 | 0.356; 0.755 | 0.567 | 0.384; 0.749 |
BDNF | 0.668 | 0.498; 0.839 | 0.418 | 0.230; 0.607 |
GDNF | 0.853 | 0.723; 0.983 | 0.392 | 0.195; 0.589 |
TIMP was applied when participants reached at least 34 weeks of postmenstrual age in order to evaluate the motor development of the preterm. The quantification of the raw score was based on the sum of the values obtained in each of the items. Raw scores were converted into percentiles according to the standardization of development curves established by the test. In order to evaluate possible associations between motor development and other variables, TIMP results were stratified into two groups: “lower than expected” (below 5th percentile) and “typical development” (above 5th percentile). Older maternal age was associated with lower than expected TIMP scores (Table
Association between clinical features and motor development results in TIMP.
Variables | Lower than expected ( |
Typical development ( |
|
---|---|---|---|
Maternal age |
27.50 (24.75; 36.0) | 24.00 (18.75; 28.0) | 0.0082 |
Gestacional age# | 31 ± 1 | 30 ± 1 | 0.3871 |
Birth weight# | 1548.8 ± 479.88 | 1388.61 ± 347.61 | 0.2741 |
1-minute Apgar |
7.00 (6.00; 8.00) | 8.00 (5.00; 8.00) | 0.9672 |
5-minute Apgar |
9.00 (7.00; 9.00) | 9.00 (9.00; 10.00) | 0.0792 |
Sex# | |||
Female | 7 (38.9) | 11 (61.1) | 0.0642 |
Male | 15 (68.2) | 7 (31.8) |
#Values expressed as mean and standard deviation for continuous variables. Number of individuals and percentages for categorical variables.
At the first time point (T0), neonates with typical motor development had higher concentrations of TNF and BDNF in the umbilical cord blood. At the other time points, plasma and urine values frequently showed similar changes. At 48 hours after birth (T1), IL‐1
Association between clinical features, plasma levels of inflammatory markers and neurotrophic factors, and motor development results in TIMP.
Variables | Motor development | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
T0 | T1 | T2 | T3 | |||||||||
Lower than |
Typical development |
|
Lower than expected |
Typical development |
|
Lower than expected |
Typical development |
|
Lower than expected |
Typical development |
|
|
IL-12p70 | 461.8550 (204.2625; 778.7525) | 445.9050 (286.1100; 541.9325) | 0.860 | 347.1500 (118.7800; 513.5800) | 345.7700 (284.8100; 406.3725) | 0.828 | 444.6450 (304.7025; 732.6200) | 414.9150 (299.1000; 513.5800) | 0.414 | 748.7100 (286.9775; 1272.8825) | 456.0650 (194.6600; 633.7150) |
|
TNF | 1.8900 (1.8150; 2.0500) | 2.0000 (1.9400; 2.3250) |
|
2.1650 (2.0000; 2.3325) | 2.2350 (1.9350; 2.7225) | 0.957 | 2.3100 (2.0500; 2.5225) | 2.1650 (2.0000; 2.8375) | 0.614 | 2.1400 (2.0000; 2.2650) | 2.0500 (1.8900; 2.2200) | 0.169 |
IL-10 | 2.2350 (1.7275; 6.4075) | 2.8700 (6.3050; 1.8650) | 0.568 | 2.0000 (1.8900; 3.1800) | 1.7400 (1.6225; 2.7975) |
|
1.8900 (1.5500; 2.6950) | 1.8650 (1.6400; 3.0075) | 0.614 | (1.7650; 4.1025) | 1.9450 (1.4625; 3.5425) | 0.540 |
IL-6 | 17.3700 (5.7900; 33.8100) | 29.6450 (12.2050; 153.0675) | 0.201 | 14.9600 (8.5025; 33.3300) | 14.6750 (8.8650; 34.8750) | 0.755 | 8.3900 (7.2800; 14.8775) | 11.8200 (7.4925; 31.0225) | 0.377 | 8.4250 (5.4400; 22.1100) | 10.0400 (5.3400; 31.1550) | 0.870 |
IL-1 |
4.1900 (3.9900; 5.0825) | 4.1850 (3.8175; 4.7150) | 0.514 | 4.1900 (4.0375; 4.2800) | 4.0400 (4.0000; 4.2300) | 0.141 | 4.1600 (4.0275; 4.3800) | 4.1150 (4.0000; 4.2000) | 0.504 | 4.0900 (3.8475; 4.5925) | 4.3400 (4.0675; 5.0250) | 0.138 |
CXCL8/IL-8 | 49.0300 (14.0350; 172.1125) | 17.1300 (12.3500; 160.4250) | 0.549 | 23.1400 (17.0550; 53.2100) | 22.5000 (14.7450; 46.9700) | 0.828 | 15.7000 (13.8100; 46.7500) | 18.2850 (15.8500; 32.4800) | 0.531 | 13.9900 (11.6300; 146.2375) | 18.8100 (12.1975; 39.1325) | 0.870 |
CXCL10/IP-10 | 93.8950 (50.8550; 134.7700) | 73.3200 (42.9325; 109.6675) | 0.414 | 62.5700 (43.1425; 103.3675) | 52.2150 (36.8950; 63.2425) | 0.165 | 40.7900 (20.2825; 79.8925) | 52.2800 (36.8600; 57.2125) | 0.369 | 59.9800 (38.9750; 123.0900) | 46.0900 (21.9350; 109.9275) | 0.765 |
CCL2/MCP-1 | 141.3000 (48.9550; 348.1200) | 138.5350 (40.8100; 265.5500) | 0.849 | 173.6600 (84.9775; 269.8575) | 92.0600 (32.5650; 173.9875) | 0.103 | 169.0100 (63.1475; 257.2900) | 80.6350 (22.8975; 131.7525) | 0.075 | 229.8450 (50.4000; 516.4825) | 156.3050 (56.1325; 409.2650) | 0.584 |
CXCL9/ MIG | 12.5300 (9.5500; 17.4075) | 10.9300 (7.4425; 21.9300) | 0.605 | 12.1650 (9.0025; 17.7350) | 9.5500 (6.5550; 15.1825) | 0.301 | 11.0600 (7.1300; 18.1100) | 12.1000 (9.2250; 14.6475) | 0.849 | 11.4100 (6.6400; 68.6350) | 13.7250 (5.6100; 18.1100) | 0.786 |
CCL5/RANTES | 4189.0300 (3490.1500; 4912.6700) | 3108.0400 (2565.0250; 4522.1000) | 0.108 | 3280.4350 (1714.4400; 5047.0700) | 3234.7900 (1013.3375; 3994.4200) | 0.341 | 4140.9550 (2707.1900; 4890.3275) | 1246.8250 (819.4700; 3959.1275) |
|
4484.2700 (4254.0700; 4923.7700) | 4429.8650 (3055.6900; 4654.5300) | 0.173 |
BDNF | 58,67050 (41,87350; 127,22950) | 136,90750 (52,52600; 291,69825) |
|
128,28300 (69,72725; 311,45300) | 175,39450 (65,41075; 252,36550) | 0.946 | 97,98350 (48,77400; 198,88925) | 211,71850 (117,52025; 400,48775) |
|
88,00000 (41,60925; 341,58475) | 141,65450 (70,52900; 303,92975) | 0.145 |
GDNF | 233,97500 (81,72825; 257,72450) | 233,97300 (199,83950; 300,28300) | 0.216 | 123,24200 (24,76400; 338,97525) | 239,41100 (146,92575; 297,91550) | 0.178 | 130,52900 (101,50500; 166,84500) | 217,26600 (151,14075; 271,96400) |
|
115,9175 (26,9508; 520,3155) | 130,4910 (30,9778; 526,7730) | 0.634 |
Median values (quartile 1; quartile 3). Mann–Whitney test. T0: umbilical cord blood; T1: 48 hours; T2: 72 hours; T3: 3 weeks after birth.
Bold characters of
Regarding molecular biomarkers, the median values at three time points (T1, T2, and T3) were used. Higher urinary concentrations of BDNF and GDNF were observed in the group with lower than expected development at the time points T2 and T3, respectively. In the group with normal development, urinary IL‐1
Association between clinical features, urinary levels of inflammatory markers and neurotrophic factors and motor development results in TIMP.
Variables | Motor development | ||||||||
---|---|---|---|---|---|---|---|---|---|
T1 | T2 | T3 | |||||||
Lower than expected |
Typical development |
|
Lower than expected |
Typical development |
|
Lower than expected |
Typical development |
|
|
IL-12p70 | 2.590 (2.400; 2.750) | 2.460 (2.393; 2.568) | 0.119 | 2.520 (2.393; 2.668) | 2.475 (2.445; 2.575) | 0.757 | 2.490 (2.370; 2.600) | 2.430 (2.355; 2.475) | 0.476 |
TNF | 1.765 (1.665; 1.955) | 1.690 (1.590; 1.803) | 0.180 | 1.640 (1.550; 1.840) | 1.765 (1.580; 1.903) | 0.251 | 1.590 (1.590; 1.715) | 1.665 (1.550; 1.840) | 0.443 |
IL-10 | 0.965 (0.880; 1.130) | 1.040 (0.930; 1.100) | 0.381 | 1.090 (0.918; 1.328) | 1.040 (0.980; 1.240) | 0.638 | 1.000 (0.980; 1.455) | 0.990 (0.930; 1.090) | 0.299 |
IL-6 | 4.140 (3.860; 5.578) | 4.235 (3.983; 6.910) | 0.667 | 4.835 (3.868; 7.105) | 4.105 (3.770; 4.780) | 0.262 | 4.520 (3.780; 11.495) | 4.050 (3.505; 8.060) | 0.199 |
IL-1 |
4.380 (4.155; 5.800) | 5.790 (5.138; 7.925) |
|
5.440 (4.520; 9.090) | 7.095 (6.015; 10.045) | 0.079 | 5.190 (4.620; 7.980) | 5.985 (4.855; 8.730) | 0.527 |
CXCL8/IL-8 | 8.415 (6.370; 32.500) | 25.710 (11.213; 60.118) |
|
8.910 (7.743; 29.780) | 33.095 (15.918; 55.230) |
|
11.360 (7.820; 40.405) | 17.570 (7.628; 53.986) | 0.925 |
CXCL10/IP-10 | 2.110 (0.680; 5.320) | 4.190 (0.680; 8.980) | 0.925 | 2.310 (0.980; 13.230) | 5.26 (2.783; 10.915) | 0.396 | 1.945 (0.680; 17.430) | 4.475 (1.875; 11.950) | 0.427 |
CCL2/MCP-1 | 24.890 (23.580; 103.805) | 40.555 (10.545; 104.098) | 0.798 | 54.230 (23.333; 123.120) | 66.320 (33.910; 96.363) | 0.510 | 46.675 (20.560; 154.975) | 45.380 (20.885; 67.118) | 0.819 |
CXCL9/ MIG | 1.035 (0.733; 1.955) | 1.140 (0.780; 1.920) | 0.581 | 1.670 (1.040; 2.408) | 1.485 (1.078; 2.130) | 0.840 | 1.220 (0.660; 5.730) | 1.250 (0.785; 8.805) | 0.677 |
CCL5/RANTES | 5.920 (4.535; 7.103) | 5.590 (4.168; 6.350) | 0.600 | 5.650 (3.525; 7.628) | 4.600 (3.700; 7.660) | 0.989 | 3.635 (3.030; 4.355) | 4.74 (4.145; 6.100) |
|
BDNF | 11.271 (0.0; 36.345) | 14.081 (3.017; 17.922) | 0.600 | 54.083 (31.736; 61.840) | 24.830 (0.0; 53.813) |
|
20.226 (13.312; 27.131) | 27.131 (16.387; 36.350) | 0.112 |
GDNF | 26.023 (17.923; 62.471) | 26.927 (20.486; 56.181) | 0.798 | 58.473 (28.887; 91.789) | 117.092 (23.158; 251.53) | 0.254 | 102.304 (52.843; 430.570) | 38.340 (18.75; 64.400) |
|
Median values (quartile 1; quartile 3). Mann–Whitney test. T1: 48 hours; T2: 72 hours; T3: 3 weeks after birth.
In this study, maternal age and urinary and plasma concentrations of inflammatory molecules and neurotrophic factors were significantly different in preterm neonates according to TIMP scores. Higher urinary levels of GDNF were found in neonates with lower than expected motor development, while IL‐1
Pregnancy disorders that lead to preterm birth may be also associated with systematic inflammation in the newborn [
The hypothesis that an inflammatory state is associated with higher incidence of brain injury is corroborated by studies showing that elevated concentrations of cytokines are associated with neuronal lesions and developmental abnormalities [
In contrast, higher IL‐1
The elevation of cytokine levels, as IL‐1
In our results, a proinflammatory response, characterized by increased levels of IL‐1
GDNF is a protective factor identified as essential for the survival and neuronal differentiation, by its action on the neuroplasticity, including the modulation of neuronal survival, axon guidance, synapse formation, and functioning in the developing nervous system [
There is some evidence for a beneficial role of neuroinflammation to the CNS. Some degree of neuroinflammation is necessary for remyelination, neuroprotection, and brain development. There are several inflammatory cytokines that regulate the production of multiple neurotrophic factors by neurons and glial cells [
In order to control the influence of confounding factors, neonates presenting acute disorders were excluded from the study. The use of corticosteroids may also interfere with this process; however, as it is recommended to prevent respiratory distress syndrome [
TIMP provides a reliable and valid measurement of the motor development that can be used for preterm neonates with 34 weeks gestational age [
The originality of this study is the evaluation of inflammatory proteins and neurotrophic factors in spot‐urine samples as a noninvasive method of collection. In addition, samples collected in time points before motor development evaluation might predict alterations in TIMP.
Measurements of inflammatory biomarkers in spot‐urine samples seem to be useful in preterm neonates. This may become a noninvasive way to follow up the inflammatory profile of preterm newborns. Whether urinary levels of IL‐1
The authors declare that there is no conflict of interest regarding the publication of this paper.