1. Relation of Vascular Endothelial Growth Factor and Insulin Like Growth factor-1 to the Development of Retinopathy in Premature Infants Prof. Dr: Enas Raafat Prof of Child Health National Research Center

2. Introduction Retinopathy of prematurity (ROP) has been identified, as a retinal vasoproliferative condition that evolves into five stages.It is initiated by retinal vascular growth retardation. Retinopathy of prematurity is one of the important causes of childhood blindness worldwide.

3. The blood vessels in the retina of premature and/or low birth weight infants are immature and underdeveloped. Following delivery, the blood vessels continue to grow and spread throughout the retina. These abnormal blood vessels are fragile and can leak, scarring the retina and pulling it out of position leading to retinal detachment. Introduction

4. Introduction The abnormal vascular changes are divided into five stages: Stage 1 ROP with demarcation line Stage 2 ROP consisting of a ridge

5. Stage 3 :consisting of a ridge with extra retinal fibrovascular proliferation Stage 4,5: refers to a partial and complete retinal detachment Introduction

6. Introduction Various risk factors to which the neonates are exposed, i.e. oxygen therapy, apnea, anemia, etc… may aggravate the insult to which the under developed or premature eye is exposed to. It has been proposed that vasculogenesis is the result of complex interactions between growth factors (cytokines like IGF-1 and VEGF) produced both locally and systemically.

7. Introduction Insuline like growth factor -1 ( IGF-1) is important for normal retinal vascular development, its deficiency is associated with lack of vascular growth, leading for subsequent proliferative ROP. Vascular endothelial growth factor( VEGF ) act as survival factor for newly formed capillaries in the developing retina and suggest an important role in the pathogenesis of human ROP.

8. Sequence of ROP Stages

9. Aim of the Study We aimed to evaluate the role of cytokine levels (IGF-1 and VEGF) in serum of premature infants as possible diagnostic markers for ROP. We also aimed to analyze other factors which may act as risk factors for occurrence and severity of ROP, like birth weight and gestational age.

10. Patients and Methods During the period from May 2008 to August 2009, 83 premature neonates who were admitted to the NICU of Almaza charity neonatal care unit in Cairo were included in this study. This work was done in corporation between, Child Health and Clinical Pathology departments (NRC) and Ophthalmology department, Cairo University.

11. Patients and Methods Inclusion Criteria: All neonates included in the study were prematures. They were classified into two groups: – Group A (cases): Premature neonates developing ROP (n = 34), they were subdivided to: 1- Mild cases (those with stages I and II ROP) 2-Severe cases (those with stages III and V ROP) – Group B (controls): Premature neonates without ROP (n = 49)

12. Patients and Methods Exclusion Criteria: – Fullterms. – Neonates with obvious congenital malformations. – Neonates with other causes for retinopathy. All babies were examined during their stay in the NICU by the attending staff and also ophthalmic examination was performed during incubation to detect ROP. A blood sample was taken from each neonate to determine serum IGF-1 and VEGF levels.

13. Patients and Methods Methodology: On admission Every patient was meticulously examined as follows: – Detailed history taking – Full clinical examination of the studied neonate – Required investigations – Ophthalmic examination: It was performed during incubation by an ophthalmologist of the NICU to detect ROP and again at 4 to 6 weeks postnatal age for follow up of premature cases.

14. Patients and Methods Methodology: After discharge All babies were seen at the follow up pediatric clinic at 4 to 6 weeks post natal age to perform the following : – Routine general and systemic examination – Ocular examination: ROP diagnosis was done by examination of the fundus with indirect ophthalmoscopy using scleral depression and documentation of the findings with wide angle digital fundus photography Retcam(2). Then recommendation for treatment was assigned to each patient

15. Patients and Methods – Cytokine analysis: Venous blood samples were collected at 4 to 6 weeks postnatal age (at the time of the ocular examination for ROP) for estimation of IGF-1 and VEGF levels in serum by ELISA techniqe. – All the cases of the study followed the rules of Medical Ethical Committee of National Research Center.

16. Results Sex distribution of cases and controls

17. Sex distribution of cases (Stage 1 and 2 versus stage 3 and 5) Results

18. Results Comparison of neonatal anthropometric data between cases and control Factor CasesControls P-value* n=34n=49 Mean± SDRangeMean± SDRange GA (week)31.6± 3.126.0-37.034.1±1.630.0-36.0 <0.001 * Birth weight (kg)1.5± 0.60.7-3.32.1± 0.61.0-3.2 <0.001 * Wt at exam. (kg)2.9± 1.51.2-7.02.9± 1.11.5-6.5 0.823 Length (cm)45.1± 2.741.0-49.047.4± 3.142.0-58.0 0.001 * HC (cm)33.7± 3.130.0-42.034.1± 2.531.0-41.0 0.819

19. Results Neonatal anthropometric data of cases (stage 1 and 2 versus stage 3 and 5)

20. Results Median duration of stay in NICU for cases and controls

21. Results Different Modes Of Oxygen Therapy

22. Results Comparison of some clinical data between cases and controls

23. Results Clinical data of cases (Stage 1 and 2 versus stage 3 and 5) Factor Stage 1&2Stage 3&5 P-value* n=16n=18 Number% % RDS1275.018100.0 0.039 * Neonatal Jaundice318.800.0 0.094

24. Results Percentage distribution of cases among stage 1, 2, 3, and 5

25. Results Comparison of cytokine serum levels between cases and controls

26. Results Cytokine levels of cases (stage 1 & 2 versus stage 3 & 5) Factor Stage 1&2 (n=16) Stage 3&5 (n=18) P-value* Mean± SD Range Mean± SD Range IGF-1 (ng/ml)13.6± 9.21.0 – 28.19.7± 8.50.6 – 24.60.324 VEGF (pg/ml)959.1± 257.9680-13501012.8± 273.6635-15000.784

27. Results Multivariate logistic regression of the effects of the variables on ROP BS.E.Sig.OR 95% C.I. for OR LowerUpper VEGF0.0190.0060.0011.021.011.03 Constant-12.5023.8810.001<0.01 B = coefficient, S.E. = standard error of the coefficient, Sig. = significance, OR = odd ratio, C.I. = confidence interval

28. Results Sensitivity and specificity values for IGF-1 and VEGF CytokineCut off95% CI * AccuracySensitivitySpecificity IGF-122 ng/ml18.5 -25.888.0%91.2%85.7% VEGF646 pg/ml565 - 71296.4%97.1%95.9% * Confidence interval

29. Results Correlation between IGF-1 and weight at examination r = 0.492

30. Results Correlation between IGF-1 and length r = 0.477

31. Results Correlation between IGF-1 and head circumference r = 0.421

32. Results Correlations between IGF-1 and data concerning oxygen administration FactorSignificance (2-tailed)Pearson Correlation Duration of stay in NICU0.2050.223 Ventilation duration0.0440.348* NCPAP duration0.2900.187 Max. FiO 2 0.6510.080 Max. PaO 2 0.259-0.199

33. Results Correlations between VEGF and neonatal clinical data FactorSignificance (2-tailed)Pearson Correlation Birth Weight0.531-0.111 Gestational Age0.154-0.250 Weight at Examination0.694-0.070 Head circumference0.797-0.047 Length0.754-0.057

34. Results Correlation between VEGF and data concerning oxygen administration FactorSignificance (2-tailed)Pearson Correlation Duration at NICU0.767-0.053 Ventilation duration0.6750.075 NCPAP duration0.719-0.064 Max. FiO 2 0.580-0.098 Max. PaO 2 0.796-0.046

35. Results Correlation between IGF-1 and VEGF P-valuesCorrelation Cases0.343-0.168 Controls0.910-0.017

36. Results Wide angle digital fundus image of the left eye for one of the cases

37. Results Wide angle digital fundus image of the left eye for the same case three days post avastin injection and laser to the retinal periphery

38. Summary Several risk factors were found to be associated with the development and severity of ROP. Data of this study suggest that LBW, small GA, oxygen therapy, long duration of incubation and critical illness of the neonate were risk factors for ROP. Regarding severity of ROP, neonates having respiratory distress syndrome or those with small head circumference were found to be at higher risk of developing severe ROP.

39. Conclusions Low IGF-1 ( 646 pg/ml) serum levels can be useful as indicators in ROP screening..For each unit (1 pg/ml) increase in VEGF value, the chance of having ROP increases by 3%. IGF-1 and VEGF were not significant risk factors for severity of ROP.

40. Conclusions Determination of IGF-1 and VEGF serum levels in the 4th week post partum provides a sufficient and reliable prognostic tool and allows the identification of a group of patients at high risk of developing ROP. IGF-1 is positively correlated with neonatal weight, head circumference, length and ventilation duration.

41. Take home Messages

42. Screening of all prematures, less than 37 weeks gestational age, and /or less than 2500 gm birth weight is importance for detection of ROP and early intervention at stages that yield good outcome. The timing of this screen should be done at the age of 4 to 6 weeks post natal age. We should not wait until the problem is solved, as this might be too late. Retinal examination should be repeated approximately every two weeks until treatment is required or retinal maturity is reached.

43. Take home Messages The time of retinal screening of premature infant is important to prevent advanced ROP which may lead to infant blindness.

44. Recommendations It is recommended that oxygen intake is to be closely monitored to avoid transient or prolonged phases of hyperoxia as recommended by many international guidelines. All premature neonates at risk, should be routinely screened at 4 weeks postnatal for serum levels of IGF- 1 and VEGF as predictive factors for ROP. Kits for IGF-1 and VEGF should be readily available for routine determinations in labs and not only for research purposes

45. Thank You