The association between βeta 2-microglobulin and bronchopulmonary dysplasia

Introduction

While the survival rates of premature infants are increasing, the rates of long-term morbidities are also growing [1]. Bronchopulmonary dysplasia (BPD) is one of the morbidities that occurs almost exclusively in preterm infants and causes several life-long problems. An intrauterine inflammatory process and many factors that cause systematic inflammatory responses such as mechanical ventilation, patent ductus arteriosus, fluid retention, and infections are considered to play a role in BPD pathogenesis [2]. The relationship between intrauterine infection or inflammation with preterm birth and BPD was revealed with the presence of proinflammatory cytokines in the umbilical cord or amniotic fluid [3].

Beta 2-Microglobulin (β2-M) is a low-molecular-weight protein found in the Class 1 Major Histocompatibility Complex structure (MHC). In systemic immune activation, cancers, autoimmune and chronic inflammatory disorders, serum and urine levels rise. Previous research has linked urinary β2-M to fetal inflammatory response and BPD in preterm newborns [4, 5]. While urinary β2-M excretion is an alternative marker for the prediction of BPD in pregnant women with chorioamnionitis, there is currently no data on the relationship between BPD and serum β2-M levels [6]. Serum β2-M can be a reliable marker of fetal and neonatal inflammatory response since it cannot pass through the placenta [7], [8], [9].

The main aim of the research was to see if there was a link between blood β2-M levels and BPD in preterm infants. Estimating the cut-off value for β2-M in predicting BPD and its connection with BPD severity were secondary goals.

Materials and methods

This study is a prospective case-control study. Before the study was performed, ethical approval was obtained from the Ethical Committee (Approval No: 29.05.2018/22), and informed consent was obtained from the guardians of the patients.

Infants whose gestational age (GA) was under 32 weeks and who were admitted to the neonatal intensive care unit (NICU) between May 2018 and November 2018 were included in the study. Patients with major congenital malformation, hydrops fetalis, lung hypoplasia, perinatal acidosis, urinary system malformations, or renal failure were excluded from the study.

As a unit policy, blood samples are taken from all newborns in the postnatal 6th hour to analyze CRP and IL-6 levels for screening for early-onset sepsis (EOS). Residual blood samples collected for sepsis screening were maintained in basic tubes with no preservatives or separation gels. Blood samples were centrifuged for 10 min at 3,000 rpm at 4 °C after waiting 30–45 min for clothing. 1 mL aliquots of serum and plasma were promptly frozen at −80 °C. After thawing, frozen materials were well mixed and centrifuged before analysis. Any samples with a hint of hemolysis were discarded. The enzyme-linked immunosorbent test (ELISA) technique (USCN Life Science Inc., Houston, TX) was used to investigate β2-M levels. For β2-M, the results were given in mg/L.

BPD was defined according to the definition of the National Institutes of Child Health and Human Development Neonatal Research Network; at a postmenstrual age (PMA) of 36 weeks, mild BPD was defined as the need for supplemental oxygen at ≥28 days but not at 36 weeks’ PMA; moderate BPD was defined as the need for supplemental oxygen at 28 days, additionally to supplemental oxygen at FiO₂ (fraction of inspired oxygen) ≤0.30 at 36 weeks’ PMA; and criteria for severe BPD, which involves the need for supplemental oxygen at 28 days and 36 weeks’ PMA, along with mechanical ventilation and/or FiO₂>0.30 [10]. In addition, they were divided into two subgroups BPD (mild, moderate, and severe) and no BPD. Retinopathy of prematurity was diagnosed according to the local classification of retinopathy of prematurity [11]. Staging of necrotizing enterocolitis (NEC) was performed according to the classification of Bell and intracranial haemorrhage was diagnosed according to the classification of Papile [12, 13]. Severe RDS was defined as ground glass appearance in chest X-rays and respiratory acidosis along with clinical signs of respiratory distress. Any of the following regularly used criteria was used to identify hemodynamically significant patent ductus arteriosus (hsPDA): In addition to clinical evidence, turbulent diastolic flow in the pulmonary artery (left to right or bidirectional shunt), left atrium/aortic root diameter ratio >1.5, ductal size >1.5 mm [14]. Early onset neonatal sepsis was defined as sepsis that occurred in the first 72 h of life, and late-onset sepsis (LOS) is defined as sepsis that occurred >72 h of life. The presence of positive cultures was described as proven sepsis. Patients who have consistent clinical and laboratory findings with sepsis but negative blood cultures were defined as suspected/clinical sepsis [15].

Maternal conditions [16] and neonatal characteristics along with preterm morbidities such as RDS, intraventricular haemorrhage (IVH), patent ductus arteriosus (PDA), NEC, BPD, ROP, and mortality was recorded. Infants who developed BPD were constituted the study group, the others who did not constituted the control group. β2-M levels were compared between the groups.

The national and the international guidelines of delivery room, RDS, and BPD management are all strictly followed by all staff and are standardized for all inborn patients [17, 18].

Results

During the study period, a total of 138 infants with GA under 32 weeks were admitted to NICU. Twenty-seven of them were excluded for not meeting the inclusion criteria, no parental consent and no obtained blood samples. Finally, data of 111 infants were analysed. Out of 111 infants, 37 died before BPD diagnosis and out of the rest 74 infants, 38 (34.2%) were diagnosed with BPD (Figure 1). Mean gestational durations were 28 ± 1.8 and 29.9 ± 1.4 weeks (p < 0.01) and mean BW were 1,086 ± 316 and 1,395 ± 348 g (p < 0.01) in the BPD group and in no BPD group, respectively. Basic characteristics of the two groups such as chorioamnionitis, preeclampsia, oligohydramnios, antenatal steroid treatment, delivery methods, and genders were similar (Table 1). While the white blood count, CRP, and IL-6 levels were similar in both groups, β2-M levels were significantly higher in BPD group (4.84 ± 1.0 and 3.79 ± 0.95 mg/L, p = 0.01) (Figure 2). β2-M levels were further corrected for GA and BW, and significantly higher β2-M levels (4.93 ± 1.15 mg/L) were found in the BPD group compared to the no BPD group (3.46 ± 1.15 mg/L) (p < 0.01) (Table 2).

Figure 1:

Research flowchart.

Figure 2:

β2-M levels of the groups.

A weak but significant correlation between β2-M level and BPD was revealed (r = 0.23, p = 0.04). Area under the ROC curve (AUC) was 0.75 (95% CI: 0.638–0.863) to predict BPD (Figure 3). β2-M cut-off value was found to be 4.75 mg/L. At this cut off value sensitivity and specificity was 68 and 73% respectively to predict β2-M levels for BPD (Figure 3).

Figure 3:

Area under the ROC curve (AUC) was 0.75 (95% CI: 0.638–0.863) to predict BPD.

A significant negative correlation was found between β2-M levels and GA (r = −0.35, p < 0.01), but not between β2-M and BW (r = −0.25, p = 0.06). Linear regression analysis was performed to test the effect of GA (beta = −0.17, p = 0.23) and BW (beta = −0.05, p = 0.7) on β2-M levels. When corrected to BW and GA, logistic regression GA and BW as covariates and a cut-off value of 4.75 mg/L for β2-M levels resulted in as, OR: 6.7 (95% CI 1.1–40.7) p: 0.039 for BPD. When further analysis performed with the defined risk factors such as β2-M levels higher than cut-off levels, hsPDA, duration of supplemental oxygen, duration of mechanical ventilation and gestational age we found that duration of mechanical ventilation was the only independent risk factor for BPD development (OR 0.92 95% CI 0.87–0.98, p = 0.016).

When we compared β2-M levels between infants with different stages of BPD, no difference was found between the groups (mild BPD: 4.33 ± 0.95, moderate BPD: 4.76 ± 1.0, and severe BPD: 4.51 ± 1.0 mg/L, p = 0.16).

Only one infant was diagnosed with culture proven EOS. A weak but significant correlation between β2-M level and suspected EOS was revealed (r = 0.29, p = 0.001). β2-M level was also significantly higher in patients with PPROM than in those without PPROM (5.25 ± 1.14 and 4.29 ± 1.07 respectively, p = 0.01).

Discussion

In this present study, our findings indicated that early β2-M levels are higher in preterm infants with BPD than infants without BPD. We could say that β2-M may be a promising marker to predict BPD in preterm infants. Our results suggest that early inflammation may have a role; however, along with the other defined risk factors, the long duration of mechanical ventilation remains the critical factor for BPD development.

Inflammation plays a key role in most preterm births [19]. Gomez et al. stated that fetal systemic inflammatory response syndrome with increasing inflammatory cytokines in blood would lead to serious morbidities [20]. It was revealed that inflammatory mediators such as IL 6, IL8, and TNF- α that were analyzed in cord blood or early postnatal period increased in the presence of chorioamnionitis and that they were also risk factors for BPD development in preterm infants [21]. Several underlying conditions in BPD development defined previously; chorioamnionitis and fetal inflammation are known to be the major contributors [22]. Even a localized inflammation makes major changes in MHC mRNA and causes the parenchymal and interstitial expression of inflammatory products in many distant and non-inflamed vital organs [23]. During the inflammatory process, Interferon γ stimulates β2-M synthesis and causes a rise in plasma β2-M levels. Serum and urine β2-M are successfully used in the diagnosis and management of inflammatory diseases, infections, lymphoproliferative disorders, and chronic autoimmune diseases in previous studies [24]. Serum β2-M can be a reliable marker of fetal and neonatal inflammatory response since it cannot pass through the placenta [7], [8], [9].

This study suggested that higher β2-M levels in early neonatal life may be associated with increased risk for BPD development. Data enlightening BPD association with serum β2-M levels in neonates are scarce. Shima et al. reported that urinary β2-M levels of ≥10 × 104 µg/gCr may predict BPD development, and elevated urinary β2-M levels at birth can be used as an alternative marker of the fetal inflammatory response [5, 6]. Similarly, in another study that included 94 infants, a strong relationship between urinary β2-M levels measured just after birth and chorioamnionitis/BPD development [5]. More currently, Shima et al. studied urinary β2-M levels of 146 infants with very low BW infants at birth and four weeks later [4]. They reported that urinary β2-M levels measured just after the birth were significantly higher in BPD infants when compared to the other two groups [4]. However, the cut-off value for β2-M level to predict the risk for BPD was not determined. The major limitations of the previous studies performed with urinary β2-M are that renal functions of the subjects included were not studied. During the inflammatory process, immune reactivation occurs, and β2-M level rises in the blood, and then it begins to rise in urine. However, in addition to inflammation, renal reabsorption may also be impaired by proximal tube damage, which may result in increased level of urine β2-M. On the contrary to inflammatory cytokines analyzed in serum, the urinary β2-M level may be a marker not only for inflammation but also for tubular dysfunction. Analysis of β2-M levels in serum samples is the major strength of this study as urine β2-M levels may be affected by renal tubular dysfunction. However, β2-M levels did not differed with the severity of BPD.

As expected the GA and BW was significantly lower in BPD group. Furthermore our results demonstrated that there is a negative correlation with β2M levels with GA. It can be speculated that already existing intrauterine inflammation which leads BPD development resulted in earlier termination of the pregnancy. Difference in β2M levels in patients with or without BPD remained when corrected by GA and BW [4]. These results can be interpreted as intrauterine inflammation itself may have an independent effect on BPD development despite the comparable CRP and IL-6 levels between the groups. Furthermore previous studies demonstrated that urinary β2-M levels are not dependent on GA [5]. It is known that preterm infants with chorioamnionitis have higher urinary β2-M levels in the first 48 h of life [6]. However to date, no association between serum β2-M and chorioamnionitis has been reported in preterm infants. In our study, only two patients had established chorioamnionitis which have similar serum β2-M levels when compared with patients who does not have chorioamnionitis, these results could be attributed to the low number of patients. However a correlation was found with serum β2-M and suspected EOS, especially serum β2-M was significantly higher in infants with PPROM and in our study that suggests an exposure to possible perinatal infection/inflammation. Kumar et al. found that antenatal steroid treatment (AST) did not change the cytokine production capacity of leukocytes examined from cord blood [25]. In our study, a relationship could not be established between AST and β2-M, and as far as we know, the data is scarce on this subject.

Gomez et al. revealed that IL-6 of >11 pg/mL in fetal blood was a marker for inflammatory response syndrome and morbidities [20]. Jobe et al. asserted that there may be a pause in fetal lung maturation due to the inflammatory cytokines [26]. According to the results of a study investigating the relationship of IL-6 in cord blood and urinary β2-M with BPD, sensitivity and negative predictive value of urinary β2-M were higher and specificity and positive predictive value were lower compared to IL-6 [27]. In our study, we evaluated IL-6 and CRP, which are the markers of inflammation, simultaneously with β2-M, but we could not reveal their relationship with BPD. IL-6 values measured in the first 6 h in preterm infants both with and without BPD in our study were higher than the cut-off value given by Gomez et al. [20].

There is also another point to discuss. This data support the hypothesis that fetal inflammation plays a key role in the development of BPD and IL-6 is an important biomarker for fetal inflammation. Unfortunately, we could not find any difference between BPD and non-BPD groups according to IL-6 levels. This can be explained by the different cascade systems of different inflammation markers or IL-6 and CRP levels may change according to the etiology of maternal infection whether it is a local agent or cause bloodstream infection or other related factors [28]. β2-M levels in the BPD group were significantly higher however no difference was found between BPD stages which suggests that accompanying morbidities such as hsPDA, sepsis, number of sepsis episodes, duration of respiratory support, and supplemental oxygen remain the major risk factors of BPD development rather than the fetal inflammation.

There are some limitations of this study. First the sample size was relatively small. Second only a single β2-M level was analysed in the early postnatal life and serial β2-M levels were not obtained so it is unclear that the difference in β2-M levels extends in later life.

Conclusions

Measuring serum β2-M levels at an early period just after the birth may be valuable in reflecting intrauterine inflammatory processes and may be useful in predicting the risk for BPD. Future studies with larger sample size and that includes analysis of other samples such as urine, amniotic fluid, bronchoalveolar lavage along with serum with some combined previously defined markers that could elucidate the fetal lung tissue inflammation/infection are warranted.