Clinical and Growth Correlates of Retinopathy of Prematurity in Preterm Infants with Surgical Necrotizing Enterocolitis and Intestinal Perforation

Abstract Objective  This study aimed to identify the clinical and growth parameters associated with retinopathy of prematurity (ROP) in infants with necrotizing enterocolitis (NEC) and spontaneous ileal perforation (SIP). Study Design  We conducted a retrospective cohort study that compared clinical data before and after NEC/SIP onset in neonates, categorizing by any ROP and severe ROP (type 1/2) status. Results  The analysis included 109 infants with surgical NEC/SIP. Sixty infants (60/109, 55%) were diagnosed with any ROP, 32/109 (29.3%) infants (22% type 1 and 7.3% type 2) with severe ROP. On univariate analysis, those with severe ROP (32/109, 39.5%) were of lower median gestational age (GA, 23.8 weeks [23.4, 24.6] vs. 27.3 [26.3, 29.0], p  < 0.001), lower median birth weight (625 g [512, 710] vs. 935 [700, 1,180], p  < 0.001) and experienced higher exposure to clinical chorioamnionitis (22.6 vs. 2.13%, p  < 0.006), and later median onset of ROP diagnosis (63.0 days [47.0, 77.2] vs. 29.0 [19.0, 41.0], p  < 0.001), received Penrose drain placement more commonly (19 [59.4%] vs. 16 [34.0%], p  = 0.04), retained less residual small bowel (70.0 cm [63.1, 90.8] vs. 90.8 [72.0, 101], p  = 0.007) following surgery, were exposed to higher FiO 2 7 days after birth ( p  = 0.001), received ventilation longer and exposed to higher FiO 2 at 2 weeks ( p  < 0.05) following NEC and developed acute kidney injury (AKI) more often (25 [86.2%] vs. 20 [46.5%], p  = 0.002) than those without ROP. Those with severe ROP had lower length, weight for length, and head circumference z scores. In an adjusted Firth's logistic regression, GA (adjusted odds ratio [aOR] = 0.51, 95% confidence interval [CI]: [0.35, 0.76]) and diagnosis at later age (aOR = 1.08, 95% CI: [1.03, 1.13]) was shown to be significantly associated with any ROP. Conclusion  Infants who develop severe ROP following surgical NEC/SIP are likely to be younger, smaller, have been exposed to more O 2 , develop AKI, and grow poorly compared with those did not develop severe ROP. Key Points Thirty percent of infants with NEC/SIP had severe ROP. Those with severe ROP had poor growth parameters before and after NEC/SIP. Risk factors based ROP prevention strategies are needed to have improved ophthalmic outcomes.


Demographic Data
The data were collected retrospectively from the electronic medical records by the research team members.We collected demographic data, including gestational age (GA), BW, sex, appropriate for GA status, race, outborn status, mode of delivery, and Apgar scores 6 at 5 minutes.We also collected maternal variables, including maternal pregnancy-induced hypertension (PIH), chorioamnionitis, and antenatal steroids.
Collected clinical information for each infant included number of days of mechanical ventilation exposure, presence of patent ductus arteriosus (PDA) and indomethacin/ibuprofen therapy for PDA treatment (before NEC), and inotrope (dopamine) use 24 hours after NEC onset.In addition, we collected information on duration, FiO 2 requirement, and mode of ventilation (invasive/noninvasive) before (birth until the day of NEC onset) and following NEC.We also collected data on the blood culture-proven sepsis at NEC onset, length of stay, and mortality.The length of stay was defined as the total duration of hospitalization from the day of admission until discharge or death due to any cause before hospital discharge.

Necrotizing Enterocolitis Data
We recorded information on the age (in days) at the time of NEC diagnosis.The diagnosis of NEC was made based on radiographic findings, including pneumatosis, portal venous gas, and pneumoperitoneum on abdominal X-ray.The frequency of surgical NEC (Bell stage III) was also collected. 17eonates who died within 48 hours after NEC onset and massive bowel necrosis found during laparotomy or autopsy were classified as having fulminant NEC.At our center, preterm infants with pneumoperitoneum who weigh less than 1 kg at NEC/SIP diagnosis and are hemodynamically unstable are treated first with a Penrose drain at the bedside but may later receive laparotomy.The timing of laparotomy after placement of the Penrose drain was based on clinical deterioration.

Necrotizing Enterocolitis Histopathological Evaluation
Hematoxylin and eosin-stained surgical resected intestinal tissue sections were evaluated for necrosis, inflammation, hemorrhage, and reparative changes.A score of 0 was assigned when the exam appeared normal, 1 for 1 to 25% necrosis/ inflammation, 2 when 25 to 50% area involved, 3 when 50 to 75% area was affected, and 4 when >75% changes were seen. 18ostoperative information such as postoperative ileus days (defined as infants being NPO [nil per os] after bowel surgery), time to reach full feeds (!120 mL/kg/d), and total parenteral nutrition days were also gathered.The surgical morbidity was classified as strictures, fistulas, wound dehiscence, surgical site infections (including abscesses), adhesions, and perforations.

Retinopathy of Prematurity Data
ROP testing was indicated if the infant was born before 31 weeks of GA, BW less than 1,500 g or after 31 weeks if considered high risk.ROP was grouped into three categories: type 1 ROP, type 2 ROP, and other ROP. 11,19Type 1 and type 2 ROP are the most severe and require treatment.Any infant with plus disease was categorized as having type 1 ROP.Plus disease indicates dilated veins and tortuous arteries in the posterior pole of the eye.Type 2 ROP is any infant having stage 3 disease.The infants having eye exam findings other than type 1 and 2 were classified as other ROP.We recorded data of infants with type 1 and type 2 ROP treated with laser photocoagulation or Avastin (bevacizumab).

Kidney Function Data
We collected all serum creatinine measurements and daily urine output data starting the day before NEC diagnosis, at NEC onset, and up to 1 week after NEC diagnosis.
[22][23][24] Bronchopulmonary Dysplasia Data Bronchopulmonary dysplasia (BPD) at 36 weeks' corrected GA was classified as mild, moderate, and severe based on the oxygen requirement at assessment. 25 We collected data on the type of steroid (hydrocortisone/dexamethasone) used during the clinical course following the NEC onset.

Growth Outcome Data
Anthropometric variables collected include weight, height, weight-for-length, head circumference, and respective z scores. 26Sex-specific Fenton growth charts were used for infants less than 50 weeks old, and gender-specific World Health Organization corrected for GA growth charts were used for infants greater than 50 weeks old.Time intervals include prior to developing NEC, during NEC treatment, post-NEC until anastomosis, after anastomosis, at 36 weeks of chronological age, and at discharge.

Brain Injury Data
Brain magnetic resonance imaging (MRI) was routinely obtained at term equivalent age or before discharge home for all preterm infants weighing less than 1,500 g at birth.Two pediatric neuroradiologists scored the MRI images independently using the scoring system of eight scales for white and gray matter injury developed by Woodward et al. 27 The categories of white matter abnormality were none (a score of 5-6), mild (a score of 7-9), moderate (a score of 10-12), and severe (a score of 13-15).

Statistical Methods
In our study, we analyzed the combined cohort of NEC/SIP and NEC separately.We evaluated all continuous variables utilizing the Mann-Whitney U test, with the results presented as median values accompanied by the interquartile range (Quartile 1; Quartile 3).In contrast, categorical variables were reported as count (n) and relative frequencies as percentages.To assess the differences among categorical variables, we employed the chi-squared test.However, when cell counts were under 5, Fisher's exact test was utilized instead.From the bivariate analyses, statistically significant variables were incorporated into the Firth's logistic regression model.In addressing missing data within our dataset, we employed listwise deletion as our primary method of missing data handling.This approach entailed excluding any preterm infants from the multivariate analysis that had missing values for any variable included in the model.By implementing listwise deletion, we ensured that analyses were conducted on complete cases only.Adjusted odds ratios (aORs) were reported as effect size along with 95% confidence interval (CI) and p-value.A two-sided p-values < 0.05 were considered as significant.Statistical analyses were performed in R Statistical Software (version 4.2.1;The R Foundation for Statistical Computing).

Oxygen Exposure and Retinopathy of Prematurity
Infants with severe ROP were exposed to higher FiO 2 at 7 days after birth (44 [30, 57] vs. 25 [21,35], p ¼ 0.001) and were intubated longer (12.5 days [7.75, 17.8]There were no statistically significant differences in the intestinal histopathology, postoperative features such as time to reach feeds and parenteral nutrition dependence, BPD, white matter and gray matter injury on brain MRI, length of stay, and mortality between infants with severe ROP and those without.The data are shown in ►Table 3.

Growth Outcomes and Severe Retinopathy of Prematurity
The preterm infants with severe (type 1 and type 2) ROP had significantly lower length and head circumference z scores before and following NEC.However, weight for length z scores were significantly lower for infants with severe ROP than the other group.The data have been summarized in ►Fig. 2 and ►Table 4.

Any Retinopathy of Prematurity
The ROP information of infants with and without surgical NEC/SIP has been summarized in ►Supplementary Table S4 (available in the online version).

Growth Outcomes
In the NEC cohort, the weight z scores and weight for length percentiles were significantly lower at 36 weeks' corrected GA for the preterm infants with severe ROP.The length z scores were significantly lower before and 4 weeks following NEC diagnosis and at the time of reanastomosis.The data have been summarized in ►Table 4.

Discussion
In our cohort of infants with surgical NEC/SIP, more than half developed ROP, but 45% did not develop any ROP.One-third of infants were diagnosed with severe ROP, of which type 1 ROP was more common.Nearly a third of the infants with severe ROP received laser treatment, and one-fifth received bevacizumab.Infants with severe ROP were smaller and younger.They were exposed to prenatal risk factors such as PIH, chorioamnionitis, and postnatal risk factors including PDA, indomethacin, AKI, more FiO 2 , and longer invasive and noninvasive mechanical ventilation duration.Additionally, infants with surgical NEC were diagnosed with severe ROP almost 6 weeks later than those without severe ROP.Infants with NEC/SIP with severe ROP also grew less well, demonstrated by lower weight z scores and linear growth before and after the NEC onset.These highly suggest systemic inflammatory response triggered by NEC, which has multiorgan system effects.
Published reports show that the prematurity and the percent and duration of oxygen exposure influence the   Clinical and Growth Correlates of ROP in NEC/SIP Infants Garg et al.
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incidence of ROP in preterm infants. 7,28The studies have demonstrated the relationship between the oxygen and the ROP with phase I (hyperoxia-induced vasoconstriction and ischemic injury) and phase II (VEGF-driven vaso proliferation) of the disease. 7n our cohort, infants with ROP were exposed to higher FiO 2 and ventilated more commonly before and after the NEC onset.In multivariate analysis, however, oxygen exposure was not a significant risk for the development of severe ROP.The possible explanation may be most likely due to three factors.First, it may be due to involvement of clinical factors other than total oxygen exposure.Second, we failed to model the in vivo oxygen saturation accurately and third small sample size in the regression models as noted by Chen et al. 28 In our cohort, the infants with severe ROP were younger (23.8 vs. 27.3weeks) and had lower BWs (625 vs. 935 g) than those without severe ROP, as reported in a previously published report. 29A study of discordant twin pairs reported that GA predicts ROP severity more accurately than BW. 30 In a prospective study from the Australian and New Zealand Neonatal Network, Darlow et al reported that prematurity was the dominant risk factor, with infants with GA of <25 weeks having 20 times greater odds of severe ROP than infants with GA of 28 weeks.BW for GA also had a "doseresponse" effect placing the more growth-restricted infants at greater risk such that infants 3rd weight percentile for GA had four times greater odds of developing severe ROP than in the 25th to 75th percentiles.Although we did not identify sex as a contributor to ROP among NEC infants, male sex was identified as a significant risk factor in at least one prior study. 31ithin our cohort, infants with severe ROP experienced poor growth, evidenced by lower length and head circumference z scores before and following the NEC/SIP onset.The infants were also exposed to chorioamnionitis, similar to a recent prospective study reporting that slower length gain and maternal chorioamnionitis were associated with delayed regression and complete retina vascularization in preterm infants. 32In this cohort, the weight z scores and weight for length percentiles were significantly lower at 36 weeks' corrected GA for the preterm infants with severe ROP.4][35] Given that poor postnatal weight gain is linked to persistently low serum IGF-1 in preterm infants, it is conceivable that the poor weight experienced in infants with NEC is a marker for retinal IGF-1 insufficiency and the resultant incomplete VEGF signaling aberrant vascular development of ROP. 36he presence and timing of NEC requiring surgery is a significant risk factor for ROP development in preterm infants. 10,11Fundora et al 11 reported that infants with early surgical NEC (8-28 days) had the highest risk of developing any ROP and severe ROP.In our cohort, infants with severe ROP had a median age of diagnosis of 23 versus 11 days for infants with no ROP, although the differences did not reach statistical significance.2][43][44] The presence of AKI following NEC/SIP onset was most likely associated with the severe ROP on bivariate analysis, which may most likely be explained due to systemic inflammation initiated secondary to NEC/SIP affecting kidneys and retina leading to multiple systemic morbidities Mechanistically, we hypothesize that severe AKI in neonates with surgical NEC may exacerbate the injury by acting as a catalyst or modifier of retinal inflammation.Further studies are needed to understand the role between severe kidney injury and ROP in preterm infants with surgical NEC.
Our study's strengths include a broad evaluation of clinical and growth parameters known or hypothesized to be associated with severe ROP.Nonetheless, our study has important limitations.First, this is a single-center experience, potentially limiting the study's generalizability.Second, sample size limits our power to detect associations between clinical factors, NEC, and ROP, which may result in type I errors.Third, in our cohort, three-fourth of the infants with surgical NEC were African American.While this is likely partly due to the demographics of Mississippi, this may also be related to adverse social determinants of health.
In conclusion, our study found that ROP developed in more than half of infants with surgical NEC/SIP, with type 1 disease being the most common.Affected infants tended to be smaller, younger, and exposed to prenatal and postnatal risk.In addition, those with severe ROP grew poorly before and after the NEC diagnosis.These findings highlight the
Clinical and Growth Correlates of ROP in NEC/SIP Infants Garg et al.This document was downloaded for personal use only.Unauthorized distribution is strictly prohibited.

Table 1
Demographics, prenatal Information, retinopathy of prematurity features, and clinical outcomes in infant with and without any retinopathy of prematurity in necrotizing enterocolitis/spontaneous ileal perforation cohort American Journal of Perinatology © 2024.Thieme.All rights reserved.Clinical and Growth Correlates of ROP in NEC/SIP Infants Garg et al.This document was downloaded for personal use only.Unauthorized distribution is strictly prohibited.

Table 1 (
Continued) Continuous data were represented as median (IQR) and categorical data were represented as frequency (percentages).p-Values were based on chi-squared test (or the Fisher's exact test when cell counts below 5) and Mann-Whitney U test.IQR represented as (Quartile 1; Quartile 3).

Table 2 (
Continued) Abbreviations: IQR, interquartile range; NEC, necrotizing enterocolitis; ROP, retinopathy of prematurity.Notes: Continuous data were represented as median (IQR) and categorical data were represented as frequency (percentages).p-Values were based on chi-squared test (or Fisher's exact test when cell counts below 5) and Mann-Whitney U test.IQR was represented as (Quartile 1; Quartile 3).

Table 3
Oxygen and ventilation data American Journal of Perinatology © 2024.Thieme.All rights reserved.Clinical and Growth Correlates of ROP in NEC/SIP Infants Garg et al.This document was downloaded for personal use only.Unauthorized distribution is strictly prohibited.

Table 4
Growth parameters in infants with and without severe retinopathy of prematurity Clinical and Growth Correlates of ROP in NEC/SIP Infants Garg et al. document was downloaded for personal use only.Unauthorized distribution is strictly prohibited.

Table 5
Association between any retinopathy of prematurity in infants with necrotizing enterocolitis/spontaneous ileal perforation and related factors using the Firth's logistic regression