Abstract
Objectives
Osteopontin (OPN) is a pro-inflammatory cytokine associated with metabolic syndrome. Extreme birth weight categories including small for gestational age (SGA), and large for gestational age (LGA) are risk factors for metabolic syndrome. However, normal levels of plasma OPN in neonates, and the relationship of OPN to fetal growth remain unknown. We evaluated the association of umbilical cord blood OPN with gestational age, birth weight, and neonatal outcomes.
Design and Methods
We performed a cross-sectional study of 261 newborns of all gestational ages beginning at week 26, and 26 adults for comparison. We collected umbilical cord blood from newborns and analyzed plasma for OPN by ELISA.
Results
Plasma OPN was significantly higher in neonates (414.65±136.72 ng/mL) compared to adults (33.37±14.66 ng/mL, p<0.001). There was an inverse correlation between OPN and gestational age (r= −0.48, p<0.0001). LGA infants had lower OPN than appropriate for gestational age (AGA) infants, but LGA was not an independent predictor of OPN in multivariate analysis. Among preterm infants, patent ductus arteriosus (PDA) was independently associated with higher OPN (OR=2.49, p=0.02).
Conclusion
Our results raise the possibility that OPN has a physiologic role in fetal growth and development, and may be a useful biomarker for PDA.
Keywords: osteopontin, newborn, cord blood, large for gestational age, patent ductus arteriosus
INTRODUCTION
Osteopontin (OPN) is a multi-functional molecule which was first isolated from bone as a highly phosphorylated extracellular matrix protein (1). OPN is related to bone health and remodeling through interactions with 1,25-dihydroxyvitamin D (1,25(OH)D) in osteoblasts. It is involved in ossification of the skeleton (2), and may play a role in early stages of osteoporosis (3). OPN also has a pro-inflammatory function and plays an important role in T helper 1 immune responses (4). Clinical studies have shown associations between circulating OPN levels and various inflammatory conditions such as rheumatoid arthritis (5), Crohn’s disease (6), and multiple sclerosis (7). Recently, elevated plasma and adipocyte OPN levels have been associated with obesity in mice and this contributed to insulin resistance and atherosclerosis (8). Conversely, OPN knock-out mice were protected from high-fat diet-induced insulin resistance (9).These studies raise the possibility that OPN links metabolic and endocrine status to inflammation and cardiovascular health, but these associations have not yet been studied.
Infants born small for gestational age (SGA) are at increased risk for adult morbidities, including metabolic syndrome and cardiovascular disease. Recent studies support the notion that both extremes of birth weight categories including small for gestational age (SGA), and large for gestational age (LGA) have increased risk for metabolic syndrome (10). The mechanisms underlying this increased risk for adult morbidities and the role of biomarkers in the newborn period is not fully understood. Circulating OPN levels were previously reported to be higher in newborns and infants than in adults in a small study (11). In addition, urine OPN levels were higher in very low birth weight infants with acute renal injury compared to infants without renal injury (12, 13). However, there are no normative data of OPN levels in umbilical cord blood, and the potential role of OPN in fetal growth and development has not yet been studied. We aimed to obtain normative data of OPN on full term and preterm newborn infants, and examine the association of cord blood OPN levels with gestational age and birth weight. Additionally we examined the relationship between cord blood levels of 25-hydroxyvitamin D level (25(OH)D) and OPN, and the relationship of OPN with prematurity-related morbidities such as bronchopulmonary dysplasia (BPD) and patent ductus arteriosus (PDA).
METHODS
Study population
This is a cross-sectional study of 261 neonates and 26 adults. Neonates included in the study were born at Brigham and Women’s Hospital (BWH) from 4/29/2010 to 9/6/2012 and were included in a prospective study on the vitamin D status among preterm and full term infant at birth. Collection of umbilical cord blood was performed from all vaginal and Cesarian deliveries when the research nurse or an investigator were available, and when the cord blood was not needed for another purpose such as cord blood banking. Patients with maternal chronic inflammatory disease, such as Crohn’s disease, ulcerative colitis, systemic lupus erythematosus (SLE), cancer, bone disease such as achondroplasia, osteogenesis imperfecta and major congenital anomalies such as congenital heart and renal anomalies were excluded.
Among the 261 infants included in analysis, 147 were full term (≥37 weeks), and 114 were preterm (35–36 weeks: 54, 32–34 weeks: 25, 29–31 weeks: 16, 26–28 weeks: 19). Our adult samples consisted of 26 subjects aged 41–55 (12 females, 14 males). Those adults were enrolled in an observational study on physical health at Beth Israel Deaconess Medical Center (BIDMC). They were mostly healthy subjects, one patient among them had previous history of stroke and type 2 diabetes. This study was reviewed and approved by the Institutional Review Board at the Brigham and Women’s Hospital (BWH) and Beth Israel Deaconess Medical Center (BIDMC). Newborns were categorized as small for gestational age (SGA : Birth weight <10th percentile), appropriate for gestational age (AGA : 10th percentile<Birth weight <90th percentile), and large for gestational age (LGA : Birth weight>90th percentile) groups based on the intrauterine growth curves derived from United States data (14).
Cord blood collection and measurement of plasma OPN and 25(OH)D
Umbilical cord blood was collected from the umbilical vein attached to the placenta at the time of delivery. Venous blood samples from 26 adults were drawn after overnight fasting at BIDMC. They were centrifuged and the plasma was divided into 0.5 milliliter aliquots, which were stored in Eppendorf tubes at −80°C until analysis. Levels of OPN were measured by ELISA by commercial kits (R&D Quantikine) with1:25 dilution of plasma sample (10 µL) at BIDMC. Intra-assay variance was <15%. The levels of umbilical cord 25(OH) D were measured by chemiluminescence immunoassay (CLIA) (DiaSorin Liaison®) at BWH.
Clinical data of newborn infants and mothers
We collected clinical information from the electronic medical records at BWH. Maternal data included were age, ethnicity, marital status, parity, multiple gestation versus singleton, insurance, smoking status, pre-pregnancy body mass index (BMI), clinical chorioamnionitis, preeclampsia, gestational diabetes, blood urea nitrogen (BUN) and creatinine (Cr). Infant data included sex, gestational age, birth weight, 1 and 5 minute Apgar scores. Birth weight Z scores were obtained using US national reference by Oken et al (15). For preterm infants, we also collected clinical information on two common morbidities of prematurity, patent ductus arteriosus (PDA) and bronchopulmonary dysplasia (BPD). PDA was diagnosed by echocardiography when the infants showed signs or symptoms of PDA such as low blood pressure, widened pulse pressure or tachypnea. BPD was diagnosed based on National Institute of Child Health and Development (NICHD) criteria (16). Diagnosis of maternal preeclampsia was made by blood pressure≥140mmHg or 90mmHg diastolic >20 weeks of gestation, and proteinuria≥0.3g protein in 24 hour urine (17). Diagnosis of gestational diabetes was made by glucose loading test with 50g glucose, and subsequent diagnostic oral glucose challenge test (OGTT) with 100g glucose. Gestational diabetes was diagnosed when two or more of the following criteria were met: the glucose level at fasting was >95 mg/dL, 1 hour glucose>180 mg/dL, 2 hour >155 mg/dL, 3 hour >140mg/dL(18).
Clinical chorioamnionitis was diagnosed with maternal fever>100.4°C with two out of four additional findings (uterine tenderness, maternal tachycardia, fetal tachycardia, foul/purulent amniotic fluid) (19). Of 261, 149 patients had pathologic review of the placenta, 106 had placental weight measured (43 placentas from multiple pregnancy were measured for only combined weight). The diagnosis of histologic chorioamnionitis was made by pathologists based on acute inflammatory change.
Statistical Analysis
One-way ANOVA test was used for continuous variables for comparison between 3 or more categories including different birth weight percentile categories (SGA, AGA, LGA). Post-hoc analysis by Bonferroni method was performed for the comparison between two groups among SGA, AGA, LGA categories. Chi square test and Fisher’s exact tests were used for categorical variables. Histogram showed a close to normal distribution of plasma OPN levels, thus, raw data of OPN levels (ng/mL) were used without mathematical transformation. Correlation analyses were performed between continuous variables including OPN, gestational age, and placental weight. We performed multiple regression analysis with OPN as an outcome variable including those variables with p<0.1 in univariate analysis.
Logistic regression analysis by forward stepwise selection method was used for prediction of LGA for full term infants, and for the prediction of PDA, BPD in the preterm population. Variables with p<0.1 in univariate analysis were included. We used SAS 9.3 (Cary, NC) for analysis.
RESULTS
Patient characteristics
Among total of 261 newborn infants, 40 (15.3%) were SGA, 200 (76.6%) were AGA, and 21 (8.0%) were LGA infants. The mean gestational age and birth weight were lower in the SGA group compared to AGA and LGA groups. Mean OPN level was significantly lower in LGA infants (323.5±136.7ng/mL) compared to AGA (425.2±140.4ng/mL) and SGA (410.0±122.7ng/mL) groups (Table 1).
Table 1.
Patients Characteristics
| All (N=261) |
SGA (N=40, 15.3 %) |
AGA (N=200, 76.6 %) |
LGA (N=21, 8.0 %) |
P | |
|---|---|---|---|---|---|
| Newborn Characteristics | |||||
| Gestational Age (weeks)*a | 37.0 (35.0, 28.6) | 36.6 (34.0, 38.3) | 37.0 (35.0, 38.6) | 39.5 (38.4, 40.1) | <0.01 |
| Birth Weight (g)*a | 2778.0 (2127.5, 3382.0) | 2232.5 (1661.3, 2547.8) | 2800.0 (2189.8, 3312.8) | 4281.0 (4185.0, 4562.0) | <0.01 |
| Birth Weight Z-score *b | −0.4 (−1.2, 0.3) | −1.7 (−1.8, −1.2) | −0.3 (−0.8, 0.2) | 1.8 (1.6, 2.2) | <0.01 |
| Birth Length (cm) *b | 47.0 (43.0, 50.0) | 44.0(41.0, 47.0) | 47.0 (43.0, 48.0) | 51.5 (51.0, 53.0) | <0.01 |
| C-section, N (%) | 204 (78.2) | 32 (80.0) | 156 (78.0) | 16 (80.0) | 0.99 |
| Multiple Gestation, N (%)* | 96 (36.8) | 19 (47.5) | 76 (38.0) | 0 (0) | <0.01 |
| Female Sex, N (%) | 136 (52.1) | 25 (62.5) | 101 (50.5) | 10 (47.6) | 0.35 |
| 1 min Apgar | 8 (7–9) | 8 (7–8) | 8 (7–9) | 8 (8–9) | 0.36 |
| 5 min Apgar | 9 (9-9) | 9 (8–9) | 9 (7–9) | 9 (9-9) | 0.12 |
| OPN (ng/mL)*a | 403.4 (321.3, 483.9) | 388.0 (327.8, 480.9) | 411.4 (327.5, 491.6) | 325.4 (269.8, 399.3) | 0.01 |
| 25(OH)D (ng/mL) | 33.3 (24.8, 42.0) | 30.9 (26.0, 37.8) | 34.7 (24.6, 43.2) | 38.1 (21.8, 40.8) | 0.24 |
| Maternal Characteristics | |||||
| Maternal Age (years) | 33.0 (30.0, 37.0) | 33.0 (29.3, 38.0) | 33.0 (29.0, 37.0) | 34.0 (32.0, 36.5) | 0.45 |
| Maternal Race | |||||
| White, N (%) | 155 (59.4) | 21 (52.5) | 117 (58.5) | 17 (81.0) | 0.10 |
| Non-White, N (%) | 102 (39.1) | 18 (47.5) | 80 (40.0) | 4 (19.0) | |
| Marital Status (married), N (%) | 187 (71.6) | 29 (72.5) | 141 (70.5) | 17 (81.0) | 0.74 |
| Maternal Insurance* | |||||
| HMO/Private, N (%) | 192 (73.6) | 30 (75.0) | 142 (71.0) | 20 (95.2) | 0.03 |
| Medicaid/Self-pay, N (%) | 69 (26.4) | 10 (25.0) | 58 (29.0) | 1 (4.8) | |
| Smoking (yes), N (%) | 5 (1.9) | 2 (5.0) | 3 (1.6) | 0 (0) | 0.22 |
| Parity (nullipara), N (%)* | 88 (33.7) | 17 (42.5) | 69 (34.5) | 2 (9.5) | 0.02 |
| Maternal BMI (Kg/m2)*a | 24.7(22.4, 29.4) | 22.2 (20.9, 25.2) | 24.7 (22.8, 29.5) | 44.6 (27.2, 49.1) | 0.02 |
| Placental weight (g)*b | 363.0 (300.0, 475.0) | 306.5 (235.0, 342.5) | 400.0 (330.0, 483.0) | 505.0 (460.0, 670.0) | <0.01 |
| Preeclampsia, N (%) | 27 (10.3) | 6 (15) | 21 (10.5) | 0 (0) | 0.19 |
| Gestational Diabetes, N (%) | 18 (6.9) | 1 (2.5) | 17 (8.5) | 0 (0) | 0.19 |
| PROM>18 hrs, N (%) | 14 (5.4) | 1 (2.5) | 13 (6.5) | 0 (0) | 0.31 |
| Clinical CAM, N (%) | 10 (3.8) | 0 (0) | 10 (5) | 0 (0) | 0.20 |
| Histologic CAM, N (%) | 16 (6.1) | 1(2.5) | 15 (7.5) | 0 (0) | 0.28 |
| Maternal BUN (mg/dL) | 9.0 (6.3, 11.0) | 10.0 (8.0, 13.0) | 9.0 (6.0, 11.0) | 8.0 (7.5, 11.0) | 0.24 |
| Maternal Cr (mg/dL) | 0.6 (0.5, 0.7) | 0.6 (0.6, 0.7) | 0.6 (0.5, 0.7) | 0.6 (0.5, 0.6) | 0.63 |
Abbreviations: SGA, small for gestational age; AGA, appropriate for gestational age; LGA, large for gestational age; OPN, osteopontin; 25(OH)D, 25-hydroxyvitamin D; HMO, health maintenance organization; BMI, body mass index; PROM, premature rupture of membrane; CAM, chorioamnionitis
Note: Variables are presented with median (interquartile range), or N (%),
p<0.05, one-way ANOVA (continuous variables), Fisher’s exact test (categorical variables)
Significant difference between LGA group and SGA/AGA groups in post-hoc Bonferroni test
Significant difference between all three groups (SGA vs AGA, AGA vs LGA, and SGA vs LGA) in post-hoc Bonferroni test
Newborns have higher circulating OPN levels than adults
We found significantly higher OPN levels in cord blood samples of neonates compared to adult plasma samples (414.65 ±136.72 Vs 33.37±14.66 ng/mL, p<0.001). We also found significantly higher cord blood OPN levels in preterm compared to term newborns (467.37±149.07 Vs 372.22 ±109.27 ng/mL, p<0.001) (Fig. 1A).
Fig. 1.
A, Comparison between plasma osteopontin (OPN) levels in adults, full term, and preterm newborn infants. One-way ANOVA test reveals a significant difference between the levels of OPN in adults, full term, and the preterm infants (p<0.001). Post-hoc Bonferroni tests reveal significant difference between adult and full term neonates (p<0.001), and between full term and preterm infants (p<0.001). Bars represent median, boxes represent 25th and 75th percentile (interquartile range), and whiskers represent 10th to 90th percentile. B, Correlation analysis (Pearson correlation coefficient) between plasma OPN and gestational age.
Cord blood OPN levels and gestational age at birth are negatively correlated
In correlation analysis, we found a significant negative linear correlation between cord blood OPN levels and gestational age at birth (r= −0.475, p <0.0001, Fig. 1B). This positive correlation remained unchanged in partial correlation analysis controlling for birth weight Z-score (Table 2B).
Table 2.
Correlation analysis of OPN, gestational age, birth weight Z-score, birth length, and placental weight
| A. Simple correlation between variables | |||||
|---|---|---|---|---|---|
| Plasma OPN (ng/mL) |
Gestational age (weeks) |
Birth weight Z-score |
Birth length (cm) |
Placental weight (g) |
|
| Gestational Age (weeks) | −0.48 ** | ||||
| Birth weight Z-score | −0.11 | 0.23** | |||
| Birth length (cm) | −0.39 ** | 0.91** | 0.38** | ||
| Placental weight (g) | −0.36 ** | 0.65** | 0.41** | 0.70 ** | |
| 25(OH)D (ng/mL) | 0.11† | −0.03 | 0.03 | −0.01 | −0.09 |
| B. Partial correlation controlling for birth weight Z-score | ||||
|---|---|---|---|---|
| Plasma OPN (ng/mL) | Gestational age (weeks) | Birth length (cm) | Placental weight (g) | |
| Gestational age (weeks) | −0.46** | |||
| Birth length (cm) | −0.37** | 0.91** | ||
| Placental weight (g) | −0.34** | 0.63** | 0.65** | |
| 25(OH)D (ng/mL) | 0.12† | −0.04 | −0.02 | −0.12 |
| C. Partial correlation controlling for gestational age | ||||
|---|---|---|---|---|
| Plasma OPN (ng/mL) | Birth weight Z-score | Birth length (cm) | Placental weight (g) | |
| Birth weight Z-score | −0.01 | |||
| Birth length (cm) | 0.12 | 0.42** | ||
| Placental weight (g) | −0.08 | 0.35** | 0.35 ** | |
| 25(OH)D (ng/mL) | 0.12† | −0.04 | 0.04 | −0.10 |
Abbreviations: OPN, osteopontin; 25(OH)D;25-hydroxyvitamin D
Pearson correlation coefficient
p<0.001,
p=0.050–0.065
Correlation of cord blood OPN and 25(OH)D
The levels of cord blood 25(OH)D showed a marginally positive correlation with cord blood OPN levels (r=0.11, p=0.06). Levels of 25(OH)D were not correlated with gestational age, birth weight, and placental weight (Table 2A). This marginally positive correlation between OPN and 25(OH)D remained unchanged in partial correlation analysis controlling for birth weight Z-score and gestational age (Table 2B, 2C).
Multiple regression analysis for factors associated with cord blood OPN levels
In univariate analysis, cord blood levels of OPN were significantly associated with gestational age, LGA status, maternal PROM>18 hours, placental weight, 1 and 5 min Apgar score (p<0.05). Plasma 25(OH)D levels (p=0.06), birth weight Z-score (p=0.07), and multiple gestation (p=0.07) showed association with OPN with marginal significance (Table 3). Since birth length showed very strong correlation with gestational age (r=0.91, p<0.001, Table 2A), we included gestational age only, not birth length in the regression models due to concern for collinearity.
Table 3.
Unadjusted univariate linear regression analysis (Outcome: OPN)
| β Coefficient | Standardized β | Standard Error | P | |
|---|---|---|---|---|
| Maternal Age (years) | 1.52 | 0.06 | 1.50 | 0.31 |
| Maternal BMI (Kg/m2) | −3.73 | −0.17 | 2.46 | 0.13 |
| Maternal Race (Non-white, Ref: White) | −8.44 | −0.03 | 17.50 | 0.63 |
| Maternal Insurance (Medicaid/ Self-pay, Ref: HMO/Private) | 2.39 | 0.01 | 19.23 | 0.90 |
| Nulliparity | 10.45 | 0.04 | 17.98 | 0.56 |
| Maternal Preeclampsia | 8.91 | 0.02 | 27.84 | 0.75 |
| Gestational Diabetes | −7.97 | −0.02 | 31.91 | 0.80 |
| Smoking during pregnancy | −24.71 | −0.03 | 61.68 | 0.69 |
| PROM >18 hr* | 127.52 | 0.21 | 36.79 | <0.001 |
| Placental weight (g)* | −0.47 | 0.36 | 0.12 | <0.001 |
| Maternal BUN (mg/dL) | −3.90 | −0.13 | 2.69 | 0.15 |
| Maternal Cr (mg/dL) | −49.89 | −0.06 | 71.99 | 0.49 |
| 25(OH)D (ng/mL) | 1.14 | 0.12 | 0.61 | 0.06 |
| Gestational Age (weeks)* | −18.25 | −0.47 | 2.10 | <0.001 |
| Birthweight Z score | −14.97 | −0.11 | 8.12 | 0.07 |
| SGA (Ref: AGA) | −5.46 | −0.14 | 23.54 | 0.82 |
| LGA (Ref: AGA) * | −99.17 | −0.20 | 30.56 | 0.001 |
| Sex, female | −0.65 | −0.002 | 16.97 | 0.97 |
| Multiple Gestation | 32.19 | 0.11 | 17.47 | 0.07 |
| C-section | −19.08 | −0.06 | 21.15 | 0.37 |
| 1-min Apgar Score* | −17.24 | −0.20 | 5.33 | 0.001 |
| 5-min Apgar Score* | −43.28 | −0.19 | 13.79 | 0.002 |
Abbreviations: OPN, osteopontin; BMI, body mass index; HMO, health maintenance organization; PROM, premature rupture of membrane; 25(OH)D, 25-hydroxyvitamin D; SGA, small for gestational age; AGA, appropriate for gestational age; LGA, large for gestational age
p<0.05, linear regression analysis
Multivariate regression analysis was performed in stepwise models as shown in table 4. Variables with p<0.1 and important demographic variables such as maternal age, parity, race, mode of delivery and infants’ sex were included in the final model. Gestational age, maternal PROM>18 hours, and 25(OH)D showed significant associations with cord blood OPN levels (Table 4). LGA was not independently associated with OPN levels in multivariate regression analysis (p=0.44, Table 4).
Table 4.
Multivariate Linear Regression Analysis (Outcome: OPN)
| Variables | Model 1 | Model 2 | Model 3 | Model 4 | Model 5 | Model 6 | Model 7 | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| β | P | β | P | β | P | β | P | β | P | β | P | β | P | |
| Gestational Age (weeks) * | −18.25 | <0.001** | −17.41 | <0.001** | −17.17 | <0.001** | −17.15 | <0.001** | −19.28 | <0.001** | −17.30 | 0.01* | −17.29 | 0.02* |
| Placental Weight (g) * | −0.10 | 0.53 | −0.08 | 0.58 | −0.08 | 0.58 | −0.06 | 0.68 | −0.13 | 0.51 | −0.08 | 0.70 | ||
| PROM >18 hrs | 90.62 | 0.05* | 90.34 | 0.05* | 92.79 | 0.04 | 105.31 | 0.02* | 117.58 | 0.01* | ||||
| LGA | −11.96 | 0.89 | −25.78 | 0.77 | −49.98 | 0.63 | −83.38 | 0.44 | ||||||
| 1-min Apgar Score | −1.22 | 0.90 | −0.92 | 0.93 | −6.21 | 0.57 | ||||||||
| 5-min Apgar Score | 30.18 | 0.24 | 30.38 | 0.24 | 20.07 | 0.48 | ||||||||
| 25(OH)D (ng/mL) * | 2.09 | 0.05* | 1.99 | 0.06 | 2.25 | 0.05* | ||||||||
| Multiple Gestation | 43.18 | 0.20 | 54.02 | 0.16 | ||||||||||
| Birth Weight Z-score | 15.46 | 0.53 | 22.99 | 0.39 | ||||||||||
| Maternal Age | 1.39 | 0.64 | ||||||||||||
| Maternal Race | 56.92 | 0.08 | ||||||||||||
| Mode of Delivery | 12.64 | 0.74 | ||||||||||||
| Nulliparity | −23.59 | 0.45 | ||||||||||||
| Infant Sex | 6.14 | 0.85 | ||||||||||||
Abbreviations: OPN, osteopontin; PROM, premature rupture of membrane; LGA, large for gestational age; 25(OH)D, 25-hydroxyvitamin D
p<0.05
p<0.001
Model 1: unadjusted regression analysis including gestational age only
Model 2: Model 1 + placental weight
Model 3: Model 2 + PROM>18 hrs
Model 4: Model 3 + LGA
Model 5: Model 4 + 1-min, and 5-min Apgar scores, 25(OH)D
Model 6: Model 6 + multiple gestation, birth weight Z-score
Model 7: Model 6 + demographic variables (maternal age, parity, maternal race, mode of delivery, and infant sex)
LGA and cord blood OPN are not independently related in full term infants
Because all LGA infants were full term in our study sample, we analyzed the association of OPN and LGA in a sub-group analysis of full term infants only (N=147). OPN was not independently associated with LGA in logistic regression analysis controlling significant factors such as gestational age, race, insurance, parity and multiple gestations.
Relationship of cord blood OPN levels and common morbidities of prematurity
We evaluated whether cord blood OPN levels are predictive of neonatal morbidities in our subgroup of preterm infants (N=114). Cord blood OPN levels were significantly higher in infants with PDA (N=11, 655.16±155.95 ng/mL) compared to infants without PDA (N=103, 449.55±133.58 ng/mL, p<0.001, Table 5A). OPN was also significantly higher among preterm infants with BPD (N=19, 570.57±169.70 ng/mL) compared to infants without BPD (N=95, 449.15±136.81 ng/mL, p=0.002).
Table 5.
| A. Comparison between PDA, and no PDA group among preterm infants | |||
|---|---|---|---|
| PDA (N=11) | No PDA (N=103) | P | |
| Gestational Age (weeks)* | 27.5 (27.2–28.2) | 35.0 (32.0–36.1) | <0.001 |
| Birth Weight (g)* | 1060 (1010–1200) | 2120 (1580–2630) | <0.001 |
| Placental Weight (g) | 250.0 (195.0–335.5) | 345.0 (275.0–405.0) | 0.06 |
| Plasma OPN (ng/mL)* | 607.9 (568.1–854.1) | 445.7 (351.6–526.9) | <0.001 |
| 25(OH)D (ng/mL) | 39.4 (22.5–65.1) | 34.2 (27.2–42.2) | 0.55 |
| Sex (female) | 4 (36.4%) | 57 (55.3%) | 0.34 |
| C-Section | 7 (63.6%) | 86 (83.5%) | 0.12 |
| Multiple Gestation | 5 (45.5%) | 62 (59.2%) | 0.52 |
| 1-min Apgar Score* | 6 (5–7) | 8 (7–8) | <0.01 |
| 5-min Apgar Score* | 8 (8-8) | 9 (8–9) | 0.02 |
| Maternal Age (years) | 35 (33–38) | 33 (30–37) | 0.14 |
| Maternal Race (Non-white) | 3 (30.0%) | 30 (29.4) | 1.00 |
| Marital Status (Married) | 7 (63.6%) | 86 (83.5) | 0.12 |
| Maternal Insurance (Medicaid/Self-pay) | 1 (9.7%) | 26 (25.2%) | 0.46 |
| Nulliparity | 6 (54.5%) | 59 (57.8%) | 1.00 |
| Preeclampsia | 1 (9.1%) | 20 (19.4%) | 0.69 |
| Gestational diabetes | 2 (1.9%) | 0 (0%) | 1.00 |
| PROM | 1 (9.1%) | 7 (6.8%) | 0.57 |
| Clinical Chorioamnionitis | 1 (9.1%) | 2 (1.9%) | 0.27 |
| Histologic Chorioamnionitis* | 4 (36.4%) | 4 (4.8%) | <0.01 |
| B. Logistic regression analysis with outcome of PDA in preterm population | ||||||
|---|---|---|---|---|---|---|
| Unadjusted OR | 95% C.I | P | Adjusted OR | 95% C.I. | P | |
| Gestational Age (weeks) | 0.508 | 0.349–0.737 | <0.001 | 0.441 | 0.218–0.894 | 0.023 |
| OPN/100 (ng/mL) | 2.363 | 1.466–3.812 | <0.001 | 2.485 | 1.146–5.390 | 0.021 |
Abbreviations: PDA, patent ductus arteriosus; OPN, osteopontin; 25(OH)D;25-hydroxyvitamin D, PROM, premature rupture of membrane; PDA, patent ductus arteriosus
Note: Data presented as median (interquartile range), or N (%)
p<0.05
Abbreviations: OR, odds ratio; C.I., confidence interval; OPN, osteopontin
Note: In logistic regression by forward stepwise selection method, variables initially included are gestational age, OPN, placental weight, histologic chorioamnionitis, Apgar score at 1, 5 min, mode of delivery, sex, parity, and birth weight.
Logistic regression analysis revealed that cord blood OPN level was an independent risk factor for PDA controlling for significant factors such as gestational age, histologic chorioamnionitis, 1-min Apgar score, placental weight, and important demographic variables including sex, multiple gestation and C-section. With every increment of gestational age by one week, adjusted odds ratio (OR) for PDA decreased by 65.9% (p=0.023), and with every 100ng/mL increment of plasma OPN level, there was 2.485 times greater odds developing PDA (p=0.021, Table 5B). In contrast, cord blood OPN level was not an independent predictor of BPD in logistic regression analysis, in which gestational age was the only significant predictor.
DISCUSSION
This is the first study to report that OPN is detectable in umbilical cord blood after 26 weeks of gestation in newborn infants, and that cord blood levels of OPN are higher in preterm infants compared to full term infants, with significant negative correlation between OPN and gestational age. This is also the first report of the association of cord blood OPN levels and PDA in a preterm population. Our study suggests that OPN potentially plays an important role in fetal growth and development through mid-pregnancy to the time of delivery. The youngest gestational age for our study population was 26 weeks. Whether OPN is detectable earlier in fetal life remains to be determined in further studies. Chen et al showed that 1,25 (OH) D increases the expression of OPN mRNA in osteoblasts in rat bone (20), but the level of OPN in relation to vitamin D in human fetal and neonatal life in the umbilical cord blood remains unknown. The exact role of OPN in regulating the vitamin D pathway in fetal life needs to be determined in further studies.
Although we did not measure maternal OPN levels in our population, two previous studies reported plasma levels of OPN in pregnant women. Schack et al reported that levels of OPN in pregnant or post-pregnant women, were not significantly different from non-pregnant adults (11). Stenczer et al reported similar plasma levels of OPN among pregnant women and non-pregnant adults. These authors also reported no difference on plasma OPN levels among pregnant women with or without preeclampsia (21). However, among a subgroup of women with preeclampsia with high fibronectin levels, suggesting significant endothelial injury, the OPN level was elevated (21). The authors suggested that this finding may indicate that plasma OPN levels reflect endothelial injury in severe preeclampsia. Given that these two prior studies reported much lower OPN plasma levels in pregnant women compared to the cord blood levels in our study, we conclude that the source of OPN in cord blood is either the fetus or the placenta and that there is very little chance that high cord blood OPN levels reflect maternal levels. Maternal preeclampsia was not associated with high OPN levels in the cord blood of newborns in our study.
OPN is known to be expressed in extravillous trophoblast and is a potential marker for stromal decidualization which regulates the invasiveness of trophoblasts (22). Further study of messenger RNA expression of OPN in placenta at different gestational ages could reveal the degree of OPN production in the placenta. Our study is also the first to report on the relationship of cord blood OPN levels and maternal PROM>18 hours which is a risk factor for intrauterine inflammation and neonatal infection. There is no previous study on the role of OPN in intrauterine inflammation and infection. Analysis of the relationship between neonatal sepsis and OPN was not performed in our study due to extremely low rate of neonatal sepsis in the study population (1 patient with Staphylococcus aureus bacteremia).
In the sub-group analysis of preterm infants, our results showed a significant association between cord blood OPN and PDA. PDA is one of the major morbidities that preterm infants can suffer from in the first weeks of life. More than 60% of preterm infants born before 28 weeks of gestation receive medical and/or surgical treatment for PDA, and PDA is known to be strongly associated with prematurity, fluid overload, and inflammatory conditions such as sepsis and chorioamnionitis (23, 24). Extremely low birth weight infants with intrauterine inflammation are known to be more likely to be non-responders to medical therapy for PDA (25). Along with the finding that the OPN level was increased in maternal PROM>18 hours, there should be further investigation of whether OPN can be a marker for inflammatory conditions in utero or in neonates.
Limitations of our study include the study population that involves a large percentage of high risk pregnancies, but we tried to minimize this limitation by excluding mothers with chronic inflammatory diseases known to be associated with high OPN levels. Our study sample consisted of 15% SGA, 76% AGA, and 8% LGA, with overrepresentation of SGA. This may be due to the fact that the Brigham and Women’s Hospital is a tertiary referral birthing center with many high risk deliveries and may limit the generalizability of our conclusion. Thus, further large scale studies in a different population are warranted. Our study design didn’t include longitudinal follow up data of study population, and it is unknown when exactly after birth the levels of OPN reach the adult level.
In conclusion, umbilical cord OPN was negatively correlated with gestational age in neonates, and was associated with cord blood 25(OH)D levels and PROM>18 hours. In preterm neonates, cord OPN levels were associated with PDA. Further longitudinal study analyzing OPN is needed for assessing the role of OPN in weight regulation and metabolism in the neonatal period and childhood.
What is already known about this subject.
Osteopontin (OPN) is a pro-inflammatory cytokine associated with metabolic syndrome in adults.
Extreme birth weight categories including small for gestational age (SGA), and large for gestational age (LGA) are risk factors for metabolic syndrome.
What this study adds
There is an inverse correlation between umbilical cord blood OPN levels and the gestational age from 26 to 42 weeks.
LGA infants have lower levels of OPN than appropriate for gestational age (AGA) infants, but LGA is not independently associated with OPN in multivariate analysis.
Among preterm infants, OPN was higher in neonates with patent ductus arteriosus (PDA), and bronchopulmonary dysplasia (BPD). PDA was independently associated with higher OPN, but BPD was not an independent factor associated with OPN in multivariate analysis.
Acknowledgements
We acknowledge Marcia Filip, Yvonne Sheldon, Elena Arons and Deirdre Greene for assistance with cord blood collection and processing, and Vanessa Gaines for data entry. We also thank Scott Weiss, M.D. and Augustus Litonjua M.D. for 25(OH)D assays, and Emily Oken, M.D. for statistical analysis and critical discussion.
This study was funded by Biomedical Research Institute at Brigham and Women's Hospital, Gerber Foundation and the William F. Milton Fund (to H.C.), Clinical Translational Science Award UL1RR025758 to Harvard University and Brigham and Women's Hospital from the National Center for Research Resources.
Abbreviations
- OPN
osteopontin
- 25(OH)D
25-hydroxyvitamin D
- PROM
premature rupture of membranes
- LGA
large for gestational age
- AGA
appropriate for gestational age
- SGA
small for gestational age
- BPD
Bronchopulmonary dysplasia
- PDA
patent ductus arteriosus
Footnotes
Conflict of Interest: None
KJ performed experiments, the first draft, and analyzed the data. HC collected umbilical cord blood samples, reviewed and edited the draft. KP made a major contribution in statistical analysis of the data, CM designed the study and directed data analysis and the interpretation as a corresponding author. All authors approved this final version of the submitted article.
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