Hyperglycemia but not Glycated Albumin, is Linked to Retinopathy of Prematurity


 Background: Retinopathy of prematurity (ROP) is a neovascular disorder of the immature retina. Neonatal hyperglycemia is a common problem in extremely preterm infants. Several studies have also reported an association between hyperglycemia and ROPPurpose: Our goal was to determine the association between hyperglycemia, glycated albumin (GlyA) and retinopathy of prematurity (ROP).Methods: Prospective study of all infants under ROP screening from March 2017 to July 2019. All demographic, clinical and laboratory data were collected. Glucose was measured at birth and every 8h for the first week and serum GlyA was evaluated at birth, 1st, 2nd and 4th weeks after birth. Reference range for GlyA was obtained according to the CLSI EP28-A3C. Univariate logistic regression was used to examine risk factors for ROP followed by multivariate regression.Results: A total of 152 infants were included in the study. Median gestational age was 30 weeks and median birth weight 1,240g. Thirty-three infants (21,7%) had ROP. Hyperglycemia was present in 24 (72,7%) infants diagnosed with any ROP versus 6 (0,05%) in those without ROP. Median GlyA at birth, 1st, 2nd and 4th and respective reference ranges were 8.50% (6.00-12.65), 8.20% (5.32-11.67), 8.00% (5.32-10.00) and 7.90% (5.30-9-00) respectively. After multivariate logistic regression, hyperglycemia but not GlyA, remained a significant risk factor for ROP overpowering the other recognized risk factors (Exp (B)28.062, 95%CI for Exp(B) 7.881 - 99.924 p <0.001)Conclusions: In our cohort, hyperglycemia but not GlyA, remained a significant risk factor for ROP overpowering the other recognized risk factors.


Background
Retinopathy of prematurity (ROP) is a neovascular disorder of the immature retina and a leading cause of preventable blindness worldwide. [1] Neonatal hyperglycemia is a common problem in extremely preterm infants [2,3] that has been linked to increased mortality and morbidity. [2,4] Several studies have also reported an association between hyperglycemia and ROP. [5][6][7][8][9][10][11][12][13][14][15][16][17] Glycation of various proteins is known to occur at higher rate in individuals with hyperglycemia. [18,19] Albumin is more sensitive to glycation than other proteins, mainly because of its high concentration and long half-life and also due to the large number of lysine and arginine residues that may be involved in the formation of early and advanced glycation products. [19 Similarly to HbA1c, glycated albumin (GlyA) can be used as a clinical marker for glycaeglycemicol. GlyA is especially useful in newborns, as high levels of fetal hemoglobin affect HbA1c measurements and re ects plasma glucose levels over a shorter period due to the shorter half-life of serum albumin when compared to erythrocytes. [20,21] The objective of this study was to determine the association between hyperglycemia, GlyA and ROP.

Study Population
This was a prospective cohort study of all preterm infants who underwent ROP screening from March 2017 to July 2019, in two neonatal intensive care units from two institutions in Portugal. The study was approved by the Ethical Review Boards of Hospital São Francisco Xavier -Centro Hospitalar de Lisboa Ocidental and Hospital Beatriz Ângelo, as well as by the National Data Protection Authority (CNPD -Comissão Nacional de Proteção de Dados) and the Ethics Committee of the Nova Medical School. It was carried out in compliance with the principles of the Declaration of Helsinki, in its latest version (Brazil, 2003).
All infants with gestational age (GA) ≤ 32 weeks, birth weight (BW) ≤ 1.500 g, or being at higher risk of ROP determined by a neonatologist were included. Infants without a known ROP outcome, and those with any ocular diseases apart from ROP were excluded.

Data Collection
For ROP screening, an initial fundus examination was performed at 32 weeks of postmenstrual age (PMA) or at 4 weeks of chronological age, whichever was later. The diagnosis of ROP and indication of treatment for ROP followed the International Classi cation of ROP Revisited [22] and the Early Treatment for ROP Study, [23] respectively. The term severe ROP included both "type 1 ROP" and "type 2 ROP" both de ned by the criteria of the Early Treatment for ROP study. [23] Demographic and ophthalmologic data, medications, laboratory results, diagnosis, procedures and maternal history were collected.
Glucose concentration was measured at birth and daily, every 8 h, during the rst week using a point-ofcare glucometer. For any glucose reading of ≤ 50 or ≥ 150 mg/dl, a serum sample was sent to the laboratory for con rmation. Infants were strati ed into two groups: a hyperglycemia group (when plasma glucose concentrations exceeded > 150 mg/dl at least once for the rst 7 days of life) and an euglycaemia group (50-150 mg/dl). Hypoglycemia was de ned as blood glucose ≤ 50 mg/dl. Serum GlyA was evaluated at birth, 1 week, 2 and 4 weeks after birth using unused blood collected for purposes other than the study. Serum GlyA was determined by an enzymatic method using albuminspeci c protease, ketoamine oxidase and albumin assay reagent (quantILab Glycated Albumin, Instrumentation Laboratory SpA -A Werfen Company, Milan, Italy). GlyA was hydrolyzed by an albuminspeci c protease, and then oxidized by ketoamine oxidase to produce hydrogen peroxide, which was measured quantitatively. Serum GlyA levels were calculated as the percentage of GlyA relative to total albumin, which was measured in the same serum sample using the bromocresol purple method. [24,25] Statistical Methods Univariate logistic regression was used to examine risk factors for ROP. In this analysis, we evaluated GA, BW, sex, small for GA status, gestational diabetes, hyperglycemia, exposure to insulin, GlyA (birth, 1st, 2nd and 4th week of life), days before rst discharge, hyaline membrane disease, bronchopulmonary, need of oxygen > 28days, days of mechanical ventilation, days of oxygen supplementation, thrombocytopenia (≤ 50 000 platelets per microliter), thrombocytosis (≥ 500 × 10 3 /L), postnatal steroids (dexamethasone), early red cell transfusion ( rst 10 days of life), total red cell transfusions, anemia (< 11 g/dl) during the rst week of life, persistent ductus arteriosus, peri and intraventricular hemorrhage, diuretics (furosemide), days of phototherapy, early and late onset sepsis, necrotizing enterocolitis (NEC) and absence of maternal milk.
Univariate analysis was followed by multivariate regression (backward conditional) using variables that were signi cant on univariate and clinically important. Each multivariable logistic regression analysis included a maximum of four independent variables always with the same dependent value, which was presence, or absence of ROP. All multivariable regression analyses were performed using a backward conditional method. We considered a p < 0.05 statistically signi cant. Reference range for GlyA was obtained according to the CLSI EP28-A3C. Data analysis was performed using the SPSSv23 (IBM®, USA).

Results
A total of 152 infants were included in the study. Median GA was 30 weeks (range, 24-36 weeks) and the median BW was 1,240 g (range, 408-2,670 g). Thirty-three infants (21,7%) developed ROP (Table 1). An interim analysis of GlyA was made for the rst 94 infants included in the study ( Table 2). Having more than half of the cohort analyzed and since GlyA was not found to be a signi cant risk factor (p > 0.05), it was decided not to pursue with its analysis to all cohort.  (Table 3). There was no statistical difference in GlyA values between infants that received prenatal corticoids vs. those who did not (p > 0.05).  is a multifactorial disease process with many associated risk factors. [17] Our aim was to determine the association between hyperglycemia, GlyA and ROP and further compare them with other known risk factors. After multivariate logistic regression, hyperglycemia but not GlyA, remained a signi cant risk factor for ROP overpowering the other recognized risk factors.
Hyperglycemia is a common metabolic disturbance affecting up to 80% of very low birth infants. [2,3] Hyperglycemia is known to be associated to oxidative stress and lead to increased resistance of vessel walls and changes in organ blood ow, thus exacerbating retinal hypoxia. [27] In an animal model of Neonatal Hyperglycemia-induced Retinopathy mimicking many aspects of retinopathy of prematurity, hyperglycemia inhibited retinal angiogenesis, induced apoptosis and retinal degeneration and led to in ammatory cytokine production. [10] Garg et al, in a retrospective case-control study of 16 infants with BW < 1000 g and ROP Stage 3 or 4 were the rst to associate increased glucose levels in the initial month of life to ROP. [17] Since then, 10 retrospective studies [5-7, 10-16] 1 prospective study [8] reported a signi cant association between hyperglycemia and ROP.
A subsequent meta-analysis, concluded that subjects with ROP had a signi cantly longer duration of hyperglycemia and an higher mean glucose level.
[28] However, when combining the adjusted odds ratio, only a statistical trend was observed on duration of hyperglycemia with ROP.
Lee et al [29] in a retrospective study of 24 548 infants with hyperglycemia, (blood glucose > 180 mg/dL), concluded that hyperglycemia alone was not associated with severe ROP. However, blood glucose > 150 mg/dL and insulin use were associated with severe ROP. In our study, hyperglycemia was de ned as blood glucose > 150 mg/dl but no infant needed insulin.
In 2019, a study by Jagla et al [30] reported that higher glycemic variability during the rst week of life was associated with treatment requiring ROP. However, this association was lost on logistic regression.
This loss might be due to real-time clinical intervention correcting the glucose level and limiting its consequences or due to the inability to detect differences due to relatively small sample size (n = 152). The same author assessed, the association between glycemic variability in the 1st week of life and type 1 ROP in a case-control study of 40 premature infants. After multiple regression, risk of type 1 ROP was only found to be associated with duration of oxygen exposure and higher glycemic variability. [31] In our cohort, the median glycaemia was 95 mg/dl in infants with ROP and 85 mg/dl in those without, supporting that the difference in median glycaemia between these two groups is not large. Nevertheless, a trend for higher glycaemia does seem to exist in babies that develop ROP. Furthermore, and in line with the studies mentioned above, we also found that the presence of any event related to hyperglycemia was more frequent in infants diagnosed with ROP versus in those without (72,7% vs. 5.0%) even after multivariate analysis.
Due to the proposed association between hyperglycemia and ROP, the authors tried to uncover if GlyA's values differed between infants with and without ROP and if so, if it would serve as clinical marker for ROP. Analogously to HbA1c, GlyA is one of the clinical markers for glycemic control and has been proven to be useful in the care of patients with neonatal diabetes mellitus. [18, 32, 33] Its main advantage over the widely used HbA1c, is that GlyA is not affected by hemoglobin's metabolism or its variants. [33] Neonatal blood contains a high proportion of fetal hemoglobin (HbF). [20,34] Additionally, preterm infants commonly undergo red blood cell transfusions which would also impair true evaluation of glycemic control with HbA1c. GlyA is not affected by the concentration of serum protein because it is expressed as a ratio to albumin, nor is affected by other proteins than albumin, and it has a high speci city as it re ects glycation products of a single protein. [32] In the particular case of infants at risk for ROP, GlyA has the advantage of re ecting plasma glucose levels over a shorter period. Because the half-life of serum albumin (14d) is shorter than that of erythrocytes, it re ects the status of glycemic control changes for 2-4 weeks. [33 20] Moreover, GlyA re ects the uctuation of plasma glucose as well as the mean plasma glucose, [32] thus having the potential of re ecting the glycemic control during the rst phase of ROP.
Koga et al. [34] have previously studied the GlyA levels in the umbilical cord blood of 5 neonates at birth. Serum median GlyA level was 9.4 ± 1.1% (slightly lower than the lower limit of normal controls for adults).
Suzuki et al. [20] tried to establish the reference intervals for GlyA in 58 healthy full-term newborn infants. Age-dependent reference values (95% CI) for GlyA in infants were between 4.9-9.4% at 4-7days, 5. Because this was a proof of concept an interim analysis of GlyA was made halfway thru the study and it was found that GlyA alone was not a signi cant risk factor and therefore it was decided not to continue this analysis.
GlyA can be altered in patients with albumin metabolism disorders. [20] None of our infants had nephrotic syndrome, or altered thyroid function, however most of them received prenatal corticoids. Two studies have reported the effect of antenatally administered corticosteroids: one study did not show an effect on whole body amino acid metabolism [35], whereas in another, infants that received prenatal corticosteroids for lung maturation tended to have a 37% increase in albumin synthesis, although not statistically signi cant.
[36] In our study there was no statistical difference in GlyA values between infants that received prenatal corticoids vs. those who did not.
Yudkoff et al reported that the relatively low serum albumin concentrations, typical of premature infants, appear to be referable to more rapid turnover of a small plasma pool rather than a diminution in the rate of albumin synthesis. [37] It is possible that the rapid turnover did not allow for su cient glycation. Other possible reason for the lack of difference between GlyA in ROP and non-ROP infants might be due to the insu cient duration of hyperglycemia.
Both data from the Continuous Glucose Monitoring in Very Preterm Infants [38] and from 2 other similar studies [3,30] monitoring the rst week of life reported that the time spent in the prede ned hyperglycemia was low (2.3-10.3% [3,30,38]). One hypothesis is that the occurrence rather than the duration of hyperglycemia might be associated with ROP.
Although not signi cant as a risk factor, with this study, we provide a reference range for GlyA at four different time points in premature infants that can be useful to assess the accurate glycemic status in these babies. Neonatal diabetes may affect gestational age and so a percentage of patients could present prematurely, or certain mutations may result in premature birth. [39] In a cohort of 750 patients with neonatal diabetes, 16% of patients with a genetic diagnosis were born prematurely. [39] There are several limitations of our study, namely the small cohort, the use of ROP rather than treatment requiring ROP (due to limited sample size) and the inclusion of only two institutions from the same geographical area. We also recorded hyperglycemic events rather than its duration, due to the inability to continuously monitor the glucose level in the entire cohort.

Conclusions
In conclusion, after multivariate analysis, the classic known risk factors -BW, need of oxygen > 28days and NEC -continued to be associated with ROP. Nevertheless, hyperglycemia remained a signi cant risk factor for ROP, overpowering the other recognized risk factors. Although not linked to an increased risk of ROP we have provided for the rst time a reference range for GlyA in premature infants, that can be used in future studies as a tool to evaluate glycemic control. Future studies should aim to prospectively investigate the effects of glucose control, preferably with continuous monitoring, in larger cohorts of premature infants, to better ascertain whether blood glucose level/variability is a speci c risk factor for ROP or rather an indirect marker re ecting the severity of the newborns' morbidity, and in turn conferring a higher risk for ROP.

Declarations
Ethics approval and consent to participate

Consent for publication
Not applicable.

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

Competing interests
The authors declare that they have no competing interests.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.