Risk Factors of Metabolic Bone Disease in Bronchopulmonary Dysplasia Premature Infants With Gestational Age Less Than 32 Weeks

Metabolic bone disease (MBD) is a complication of multifactorial aetiology in preterm infants. Several risk factors have been identied in general. Bronchopulmonary Dysplasia (BPD) infants present an increased incidence of MBD, but it is unknown which factors contribute to this. The aim of this study was to determine the risk factors for developing MBD in BPD infants. A retrospective review of the medical records of BPD infants admitted to the Neonatal Intensive Care Unit (NICU) at Zhangzhou Hospital between Jun 2016 and May 2020. BPD infants with MBD were identied, two contemporaneous without MBD matched by gestational age and gender were randomly selected as control infants for each case of MBD. The association between putative risk factors and MBD was estimated with ORs and 95% CIs. A P-value threshold ≤ 0.2 was used in univariate analysis for inclusion into a multivariate (adjusted) model with a P-value of < 0.05 as statistically signicant.


Introduction
Metabolic Bone Disease (MBD) of prematurity is a multifactorial disorder characterized by low bone mass and demineralization of bone tissue posing a consequent increase in bone fragility and long-term reduced linear growth and childhood or even adult height, which is commonly observed in very low birth weight (VLBW, <1,500 g), especially in extremely low birth weight (ELBW, <1,000 g) infants with a reported incidence of about 16%-40% [1]. BPD infants have a higher incidence (60% vs 34%, compared with infants without BPD) of MBD in infants 1,250 g [2]. Among infants born 1,500 g suffered from severe BPD, about one third developed severe MBD [3].
Several factors associated with increased risk of MBD had been identi ed. Costa R et al found that the majority of MBD infants were male and presented lower gestational age, lower birth weight, and prolonged duration of parenteral nutrition [4]. Chen W et al found that birth at <30 weeks of gestation, vitamin D supplementation at >14 days of age, and achievement of total enteral nutrition (TEN) beyond 28 days of age were independent risk factors for MBD in infants 34 weeks gestational age [5]. Montaner Ramón A et al pointed only restricted fetal growth was independently associated with the development of severe MBD (OR 9.65, 95% CI 3.48-26.76) in infants 32 weeks gestational age and/or weight 1,500 g [6], and Ukarapong S et al indicated that only cholestasis remained signi cantly risk factors (OR 9.6, 95% CI 2.1-45.3) in infants <30 weeks gestational age and/or weight <1, 000 g [7]. Although BPD infants presented an increased risk for developing MBD, previous studies did not evaluate the risk factors that would be associated with MBD in BPD infants.
The purpose of this study was to assess the risk factors for MBD in BPD infants, which might contribute to identify high-risk individuals and propose further recommendations for management of the comorbidities.

Study population and setting
We performed a retrospective review of the medical records of BPD infants admitted to the Neonatal Intensive Care Unit (NICU) at Zhangzhou Hospital between Jun 2016 and May 2020. The study was approved by the Institutional Review Board of the Zhangzhou Hospital, with waiver of informed consent.
Case with MBD were identi ed as case group. Inclusion criteria consisted of the following: (1) gestational age 32 weeks, (2) survival up to 36 weeks post-menstrual age (PMA), (3) diagnosis of BPD according to National Institutes of Health (NIH) that de ned as requirement of oxygen support (>21%) for at least 28 days and a subsequent assessment at 36 weeks PMA or discharge, whichever comes rst. At the time of assessment, infants with no oxygen requirement were classi ed as having mild BPD, infants requiring <30% oxygen were classi ed as having moderate BPD and infants with a need for positive pressure ventilation/continuous positive pressure (PPV/CPAP) and/or oxygen requirement ≥30% were classi ed as having severe BPD [8], and (4) diagnosis of MBD that de ned as peak serum ALP higher than 900 U/L and serum phosphate level lower than 1.8 mmol/L, which yielded a sensitivity of 100% at a speci city of 70%, with or without radiographic changes of long bones [9]. Exclusion criteria included the following: (1) gestational age≥32 weeks, (2) presence of signi cant congenital anomalies, (3) death before 36 weeks PMA. For each MBD case, two contemporaneous with BPD but without MBD matched at equivalent gestational age and gender (male: female=1:1) were randomly selected.
Clinical data on demographics and putative risk factors for MBD were collected, including birth weight, gender, use of antenatal steroids, histologic chorioamnionitis (HCA), fetal growth restriction (FGR), maternal hypertensive disorders without FGR, prolonged rupture of membranes (PROM), type of feeding, initiation of oral vitamin D supplementation, feeding volume at the end of the 4 th week after birth, postnatal dexamethasone application, patent ductus arteriosus (PDA), cholestasis, use of mechanical ventilation, late onset sepsis (LOS), thyroid function tests and platelet (PLT) count.
FGR was de ned as antenatal diagnosis by any 2 of the following: estimated fetal weight below the 10th percentile according to the care provider reference curve, abnormal fetal Doppler ndings, growth arrest, or maternal hypertensive disorders. PROM was de ned as rupture of the membranes occurring earlier than 24 h before delivery [10]. HCA was de ned as the in ltration of neutrophils in any of the following structures: placental disc, the chorioamniotic membranes, and the umbilical cord [11]. Cholestasis was de ned as direct bilirubin >2 mg/dL [12]. Maternal hypertensive disorder was de ned as systolic blood pressure ≥ 140 mmHg and (or) diastolic blood pressure ≥ 90 mmHg [13]. PDA was diagnosed with echocardiography con rmation. Thyroid-stimulating hormone (TSH) = 10 mU/L was used as the cutoff level for hypothyroidism [14] and PLT 100×10^5/L was considered to be thrombocytopenia [15]. LOS was diagnosed with clinical suspicion and evaluated by microbial culture or two non-speci c blood indicators occurring after 72 h of birth [16,17].
In our NICU, we started parenteral nutrition in the rst hours after birth and introduced enteral feeding with breast milk as soon as the infant reached a tolerated respiratory rate without hemodynamic instability. The amount increased gradually with a rate of 20-30 mL/kg/day without signs of feeding intolerance.
Oral vitamin D (800-1000 IU) supplementation was started when the amount of breast milk feed reaches 50 mL/kg/day, and liquid human milk forti cation was added when the amount of breast milk feed reaches 80 mL/kg/day. Premature formula was also used if the amount of breast milk is insu cient.

Statistical Analyses
Data were analyzed using SPSS version 26. Dichotomous or categorical variables were compared between cases and controls with the use of χ2 analysis or the Fisher exact test or z-test. Continuous variables were compared between groups via the Mann-Whitney U test. A risk model was derived via logistic regression, with 95% CIs calculated for ORs using the forward LR method. A P-value of ≤ 0.2 was used in univariate analysis for inclusion of putative risk factors into the multivariate (adjusted) model. A P-value of < 0.05 was considered statistically signi cant [16].

Results
During the study period, a total of 156 infants with BPD were enrolled with 52 cases of MBD and 104 controls. Demographic and clinical characteristics among cases with MBD and controls was shown in

Discussion
The growing advances in the intensive care of preterm infants have led to a decrease in mortality, but cause more frequent morbidity such as MBD and BPD, etc. Previous studies have proved that MBD is inversely associated with gestational age [4,5], hence we matched the gestational age equal to the cases when selecting the controls to allow evaluating other putative factors in homogeneous groups. This study provides novel data on risk factors for MBD in BPD infants. FGR, ELBW (birth weight 1000 g), feeding volume 80 mL/kg/day at the end of the 4 th week after birth, cholestasis and LOS each served as statistically signi cant, independent risk factors, on the basis of their associated ORs from multiple logistic regression.
The association between FGR and MBD in BPD infants was con rmed in our study which also in accordance with other data showing that FGR was a risk factor for BPD published [8]. This early life factor seemed to ease MBD development in a programmed process and might be used as an early predictive indicator for screening of MBD. FGR commonly coexists with preeclampsia at gestation, which results in placental abnormalities that deteriorates placental transfer of calcium, magnesium and phosphorus [19], and thus contribute to MBD. Furthermore, our study showed that maternal hypertensive disorder without FGR was not associated with MBD, indicating the disrupted placental as key pathogenesis. In addition to FGR, ELBW was also an independent risk factor for MBD in BPD infants of equal gestational age in our study samples, revealing an inverse association between birth weight and MBD, which was found even in ELBWs exclusively by Viswanathan S et al [20].
Feeding problems are almost inevitable in the very preterm infants. Generally, it is not di cult to introduce enteral feeding but hard to reach total enteral feeding owing to uncomfortable abdominal distention or signs of necrotizing enterocolitis (NEC), which may disrupt the feeding schedule. Prolonged PN were demonstrated to associated with MBD by several researches elsewhere [21]. Similarly our study revealed that an enteral feeding volume 80 mL/kg/d at the end of the 4 th week after birth remarkably increased the risk for MBD in BPD infants. This may be explained by the following reasons. First, the small breastfed volume did not allow the introduce of breast milk forti er in our NICU. Breast milk provides inadequate protein and mineral content which cannot guarantee enough calcium and phosphate intake for preterm infants. Second, the su cient feeding volume still require PN supplement which pose the possibility of aluminum contamination and the risk of mineral precipitation in the solution due to the small volumes [22]. It has been demonstrated that newborns fed exclusively with breast milk showed lower levels of phosphate [23] and forti ed breast milk feeding signi cantly increase bone mineral density (BMD) values via linear regression analysis [24]. Therefore, an earlier begin of forti cation of the breast milk begins might be taken into consideration in breast-fed preterm infants with high risk for BPD, as appropriate. Some studies showed that vitamin D supplementation at >14 days of age was also associated with MBD, however, our study did not consistent with it. A possible explanation is that it is the blood levels of 25(OH)D3 that is directly associated with MBD [6]. A usual intake dose of 800-1000 IU/d for preterm infants with BPD did not achieve the protective level, which is probable due to fat-soluble vitamin de ciency caused by the coexisting cholestasis [25]. Simultaneously, cholestasis was also a risk factor for MBD in BPD infants con rmed by our study and other researches [21]. In other words, these infants may be at a greater demand for vitamin D supplementation.
LOS remains an important problem and are associated with complications of preterm infants including BPD [27]. Jensen EA et al found that blood culture con rmed sepsis were associated with increased odds of severe MBD in infants with severe BPD [28]. Our study reenforced the association between LOS and MBD in BPD infants. It has been acknowledged that important interactions occur between immune and skeletal systems [29]. Lipopolysaccharide exposures could result in bone loss [30], which might be due to the activation of B and T cells that potentially regulate bone resorption [29]. Approaches, such as a strict hygiene protocol and the minimization of invasive procedures in NICUs should be employed in prevention of LOS in order to reduce MBD.
The strengths of this study are as follows, rst, we enrolled only infants with BPD and matched gestational age to ensure the comparison in homogeneous groups, second, the monocentric study guaranteed that the enrollments were managed strictly under the same perinatal practices including diagnosis and treatments. Still, there are some limitations. MBD is a multifactorial disease. The small sample size may mask some risk factors that could not be displayed. However, we determine some risk factors that could be interpreted. The results in our study revealed that MBD in BPD infants shared some same risk factors with preterm infants in general.

Conclusion
In gestational age homogeneous BPD infants, fetal growth restriction, ELBW, feeding volume 80 mL/kg/day at the end of the 4 th week after birth and LOS are signi cant risk factors for MBD. Given the observational nature of this study, further longitudinal/prospective studies are required to validate these ndings.

Authors' contributions
Xu LP had primary responsibility for the study design and protocol development. Chen WW performed the nal data analyses and contributed to the writing of the manuscript. Zhang ZH was involved in the data collection. Dai SZ was involved in the table drawing. All authors read and approved the nal manuscript.

Funding
The study was supported by Startup Fund for scienti c research of Fujian Medical University (no.2019QH1270).

Ethics approval and consent to participate
The study was approved by the Institutional Review Board of the Zhangzhou Hospital. The study is based on register data, and individual informed consent from each participant is not required by waiver from the ethical committee and national guidelines.

Consent for publication
Not applicable.

Competing interests
None declared.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.