Analysis of pathogenesis and risk factors associated with retinopathy of prematurity

，

(1.School of Medicine，South East University，Nanjing 210009，China; 2.Department of Pediatrics，Zhongda Hospital，South East University，Nanjing 210009，China)

1 Introduction

ROP is a leading cause of childhood blindness， accounts for up to 10% of childhood blindness in developed countries[1]and is associated with significant sequelae， including retinal detachment， ametropias， refractive errors， strabismus， and disorders of color discrimin-ation[2- 3]. The proportion of blindness as a result of ROP differs significantly across different geographical locations， and this might be attributed to varying levels of neonatal care and the availability of effective screening and treatment programs[4]. Inspite of the improvements in neonatal care and treatment， the incidence of ROP remains static， if not on the increasing trend.ROP is the major cause of vision loss in premature infants， especially among low birth weight infants.

ROP is a disorder of a developing retina of premature infants.ROP， formerly known as retrolental fibroplasias， was first described by Terry，who connected ROP with premature birth[5]. It has been classified into 6 stages， according to the severity， location into zone Ⅰ- Ⅲ and by the presence of pre- plus and plus disease.In stage 0， there is immature retinal vasculature with no clear demarcation of vascularized and nonvascularized retina; stage 1， a demarcation line can be found separating the avascular retina anteriorly from the vascularized retina posteriorly， with abnormal branching of the immediately posterior small vessels; in stage 2， there is a retinal ridge which remains intraretinal; stage 3 shows a ridge with extraretinal fibrovascular proliferation; in stage 4， there is partial retinal detachment; and in stage 5， total retinal detachment can be seen. In plus disease there is arteriolar tortuosity and venous engorgement of the posterior pole， whereas pre- plus disease diagnosed when the vascular abnormalities of the posterior pole are insufficient for the diagnosis of plus disease but demonstrate more arteriolar tortuosity and more venular dilatation than normal[6]. Studies conducted over the past few decades and reports of ROP associated with oxidative stress， various angiogenic and maternal factors， inflammatory mediators and infection have renewed interest in exploring further into ROP. This review discusses the implications of various risk factors on the pathogenesis of ROP and the need to delve deep into it to pave way for novel or adjunctive management．

2 Epidemiology

The advancements in neonatal management over the last forty years have brought about a significant increase in the survival rates of both the ELBW infants and VLBW infants. It has increased from 5% to 65% in the former one and from 35% to 90% in the latter. This increase in survival of neonates has led to increase in number of diagnosed cases of ROP over time. The incidence of ROP has varied over time， there has been various reports indicating decrease， no change or increase in incidence of ROP[7]. The Cryotherapy for Retinopathy of Prematurity Study(CRYO- ROP Study)was the pioneer analysis of the incidence of this disease.They reported a ROP incidence of 65.8% in infants with birth weight less than 1 251 g[8]. Later ROP incidence of 68% was reported by Early Treatment for Retinopathy of Prematurity Study(ENTROP study). This study also observed a group of premature children with birth weight <1 251 g and found that incidence of ROP was inversely proportional to the gestational age and birth weight[9]. Varying geographical locations also seem to play a role in the incidence of ROP. In southern Taiwan， ROP was identified in 37.8%， with ROP diagnosed as stage 1， 2， 3， 4， and 5 in 12.1%，7.2%， 16.1%， 2.2%， and 0.2% infants， respectively[10]where as in India incidence of any ROP was 21.6% and severe ROP was 6.7%[11]. A study conducted in Canada， showed incidence of 40.4% for ROP， 9.2% for severe ROP， and 5.67% for ROP requiring laser treatment[12]. However， this significant variation in incidence of ROP among different geographical locations may be due to the different methods applied in management and screening and the level of neonatal care.

3 Pathophysiology

With reference to the mechanisms involved in the pathogenesis of ROP， we have come a long way since the 1940s when Terry described it for the first time as retrolental fibroplasias.Earlier studies done on animal models in particular must be reconsidered owing to the fact that there has been significant advancement in neonatal care over the years especially because the oxygen levels used then differ considerably from those currently used in preterm infants.

Retinal vessels initially originates from optic disc approximately at 4thmonth of gestation and progresses outwards towards ora seratta just before term， therefore preterms have poorly vascularized retina with avascular peripheral zones. Normally in utero， the blood is only 70% saturated compared to 100% in full term infants in room air. The normal PaO2 in utero is 30 mm Hg， while a normal infant breathing room air will have a PaO2 of 60-100 mm Hg. This change is oxygen tension to the developing retina leads to progression of ROP. ROP is a biphasic disease， with phase one consisting of delay in physiologic retinal vascular development in a hyperoxic environment， which occurs from birth to postmenstrual age approximately 30-32 weeks where as phase two which begins around 32-34 weeks postmenstrual age， with abnormal neovascularization of retinal vessels in response to hypoxemia. Therefore， after birth premature infants are exposed to hyperoxic extra- uterine environment as well as supplemental oxygen are mostly given to these infants are thought to be responsible for this process. In an ELEGAN study they found that repeatedly high blood concentrations of oxygen on least 2 of the first 3 postnatal days was associated with increased risk of severe ROP[13]. This study showed that severe retinopathy of prematurity was more likely to develop in infants with a PaO2in the highest quartile as compared with the lowest quartile. In the second phase of Retinopathy of prematurity， upon return to room air the non- perfused portions of retina become hypoxic， thereby inducing the expression of angiogenic factors and resulting in retinal neovascularization[14].

Since most mammals complete their retinal vascularization postnatally， animal models were developed to test the role of stresses in preterm infants on the pathogenesis of retinopathy of prematurity. Much of our understanding regarding the underlying mechanism of the disease process comes from the use of animal models. The oxygen- induced retinopathy(OIR) in rodents has been a valuable tool to researchers studying ischemic retinopathies， providing substantial insight into these conditions， since they can be used to study the pathways involved in angiogenesis[15]. In these models neonatal mice were exposed to 75% oxygen from 7thpostnatal day to 12th， under this hyperoxic environment cessation of normal radial vessel growth occurs similar to phase one ROP and these mice when returned to room air， the non perfused portion of retina becomes hypoxic which leads to expression of angiogenic factors that causes retinal neovascularization， similar to phase two ROP. Although these studies have contributed profoundly on understanding the disesase process， there are limitations when comparing animal models to human.In most of the OIR studies the rodents used were not premature. First， in a recent study in which they investigated the effect of preterm birth on retinal vascularization using the neonatal rat， they found that preterm pups had significantly larger avascular retinal areas than term rats on the various postnatal days.After exposure to cyclic oxygen， preterm pups demonstrated significantly larger avascular and neovascular areas compared with term rats and residual retinopathy of preterm pups was greater than that of term pups[16]. Secondly， in OIR studies mice were exposed to higher level of PaO2， up to 500mg or more[17]， where as in ELEGAN study they showed that severe retinopathy of prematurity was more likely to develop in infants with a PaO2in the highest quartile. However， the median PaO2was approximately 100 mm Hg on day 1 for all stages of retinopathy of prematurity， no infant had a PaO2level as high as 400 mmHg[13]. Thirdly， in preterm the oxygen level fluctuates on minute to minute basis， where as in most mouse model of OIR， oxygen level is constant[18]. Therefore various researches on animal models have been helpful in understanding the disease process but there are certain limitations when comparing these models with humans．

4 Role of various angiogenic factors

Various angiogenic factors such as VEGF，IL- GF and erythropoietin have been implicated in the patho-genesis of ROP. VEGF is essential for the growth of retinal vasculature， normally in response to the hypoxia. The expression of VEGF stimulates blood vessel growth whereas in phase one of ROP， hyperoxia suppresses VEGF expression， resulting in the loss of the physiological wave of VEGF to the growing vascular front. It is also found that serum VEGF levels at birth are lower in infants who later develop ROP. Following oxygen- induced vessel loss and subsequent hypoxia in phase two of ROP， VEGF expre-ssion is increased in retina， resulting in pathological neovascularization[19].

Prematurity is considered a major risk factor for ROP and therefore， factors involved in growth and development (IGF， GH)possibly have something to do in retinal neovascularization. IGF- 1 is mostly supplied by placenta and amniotic fluid to the fetus. The serum concentration of IGF- 1 increases with gestational age and correlates with fetal size， therefore without the placental and amniotic fluid supply， extrauterine growth restriction and delayed physiologic retinal vascularization can occur. The role of IGF- 1 in phase one of ROP is supported by various clinical studies. The mean serum levels of IGF- 1 in age- matched premature babies are directly correlated with the severity of clinical ROP and interestingly may also lead to abnormal brain development[20]. IGF- 1 is also required for maximum VEGF activation of vascular endothelial cell proliferation and survival pathways.IGF- 1 levels are deficient after premature birth， setting the stage for retinal vascular loss and ROP， restoration of IGF- 1 to levels found in utero may help prevent ROP[21].Some studies show that infants with extrauterine growth restriction and low weight gain are prone to severe retinopathy of prematurity whereas administration of IGF- 1 in growth-restricted mice reduced oxygen induced retinopathy in one study[22]. These findings indicate the possible role of IGF- 1 in reducing severe retinopathy of prematurity. IGF- 1 regulates retinal neovascularization at least in part through control of VEGF activation of p44/42 MAPK， establishing a hierarchical relationship between IGF- 1 and VEGF receptors. So， IGF- 1 acts as a permissive factor for VEGF- dependent endothelial growth and survival[23].

Erythropoietin(Epo)， red blood cell stimulator， is also a promoter of vascular endothelial cell proliferation and angiogenesis. Early administration of erythropoietin reduced phase 1 avascularization in both mouse and rat models of oxygen- induced retinopathy. In murine models of oxygen- induced retinopathy， inhibition of Epo led to inhibition of retinal neovascularizationinvivoand inhibition of retinal endothelial cell proliferationinvitro[24]. An association between Epo and severe oxygen induced ROP[25]have been shown by various studies. We suggest that further studies are required regarding the role of Epo and timing of its use in ROP.

Reactive oxygen species(ROS) activate signaling pathways that result in either physiologic or pathologic effects such as apoptosis and angiogenesis. An increased generation ROS in response to hyperoxia， reperfusion， and/or infection has been associated with development of ROP. The balance between ROS and antioxidant may be disturbed in premature infants since the retinal antioxidant reserve is not sufficient in premature infants to provide protection against ROS[26].The efficacy of antioxidants such as vitamin E， N- acetylcysteine， and lutein have been inconclusive or have shown unacceptable side effects in infants with retinopathy of prematurity as yet[27]. Infants born prematurely have only 10% of adult levels of retinal vitamin E. One study suggested that in very low birth weight infants vitamin E increased the risk of sepsis， and reduced the risk of severe retinopathy and blindness. So，routine of vitamin E supplementation is not advised[28].In preterm infants with early onset sepsis，there is a relationship between high plasma levels of cytokines IL- 6，IL- 8，and TNF- α in the first days of life with the development of ROP requiring treatment in the later stages[29]. Levels of circulating pro- inflammatory cytokines are elevated at multiple time- points after birth in preterm infants who later develop ROP when compared to controls[30]. These definitely give a clear picture that the increased systemic levels of cytokines are associated with an increased risk for ROP．

5 Risk Factors Associated with ROP

5.1 ROP and Blood Gases

The association of prolonged exposure to high oxygen concentration and retrolental fibroplasia， currently known as ROP， was discovered almost half a century ago.This point in time， the most effective means to diminish the rates of ROP largely involve restricting tissue oxygenation by maintaining lower levels of hemoglobin saturation with oxygen in infants born prematurely since oxygen exposure in the first few weeks of life increases the likelihood of ROP occurrence[31]but some studies have also implicated that high oxygen supplementation during the second phase of ROP has protective effects[32]. Since second phase of ROP begins around 32- 34 post menstrual age(PMA)， supplemental oxygen at this PMA might improve retinal oxygenation and down- regulate retinal neovascularization in phase II of ROP. Hypercarbia and acidosis also increase retinal neovascularization in animal models[33- 34]. In ELEGAN study infants exposed to PCO2values in the highest quartile for their GA and pH in the lowest quartile was associated with an increased risk of severe ROP[13]. Infants in mechanical ventilation who are exposed to high oxygen pressure for a long period of time may develop ROP[35]. Therefore even with the monitored management of oxygen， carbon dioxide and pH，the incidence of ROP remains unchanged. More studies are needed to better characterize the relationships of oxygenation， ventilation and acidosis with the risk of ROP development．

5.2 ROP and Infection

Infection and sepsis are frequently encountered problems in preterm and are associated with significant morbidity， neonatal complications， prolonged hospitali-zation， and death. Sepsis in neonates is considered an independent risk factor for ROP[36]. In ELEGAN study， it was observed that late neonatal bacteremia was an independent risk factor for ROP[37].But， in a large cohort study， infants who developed early onset sepsis also had an increased risk for severe ROP[38]. Candida species release pro- inflammatory cytokines， which interacts with vascular endothelial cells causing injury to the developing retinal blood vessels， hence， these species， has been independently associated with increased severity of ROP. Systematic review and meta- analysis of eight studies found that systemic fungal infection in very low birth weight infants was significantly associated with ROP and severe ROP[39]. Control of infection is mandatory in order to avoid development to ROP in preterm infants， whether the infection is early onset or late onset.

5.3 ROP and Maternal Factors

A myriad array of maternal factors， including body mass index， in vitro fertilization， placenta abruption， first partum， delivery method [vaginal or cesarean section]， premature rupture of membrane and pre- elampsia have been explored in the past and have been shown to have some role in the development of ROP. Chorioamniotis[40]and maternal inflammation[41]have also been implicated in the development of ROP by decreasing the level of IGF- 1 in preterm infants. Clinical chorioamnionitis and elevated maternal WBC count， but not histologic chorioamnionitis， were significantly and independently associated with ROP[42]. Multiple gestation as an independent risk factor and older maternal age as a risk factor for the development of ROP in premature babies[43- 44]have also been noted. Variation in the incidence of ROP among various maternal ethnic groups has also been reported. Infants born to Asian mothers were at increased risk of development of ROP requiring treatment compared to white or black counterparts[45]. Maternal pre-eclampsia was found to be associated with increased ROP development risk in premature infants[46]， whereas some have noted that Preeclampsia， but not gestational hypertension， was associated with a reduced risk of ROP in preterm births[47]. Many placental factors such as inflammatory cytokines，corticotropin- releasing hormone， free radical species and activin A are increased in preeclampsia and these factors can alter vascular development and may promote development of ROP[48]. Although various maternal factors have been implicated as risk factors for ROP we suggest that further larger studies are needed to conclude these findings．

5.4 Various Other Factors and ROP

The association of ROP with various factors including low birth weight， young gestational age， non- black race， birth outside of a study center， oxygen exposure， resp-iratory distress syndrome， apnea， erythrocyte transfusions， sepsis， intraventricular hemorrhage， prolonged parenteral nutrition， methylxanthine administration， treatment with indomethacin， renal insufficiency， and poor postnatal weight gain have been there for a long time. Among these， low gestational age， low birth weight， oxygen exposure， blood transfusion， total paren-teral nutrition， necrotizing enterocolitis， intraventricular hemorrhage， bronchopulmonary dysplasia and neonatal sepsis are considered the more important risk factors[49]. Minghua et al suggested that neonatal sepsis， oxygen exposure， and low gestational age are not only indep-endently associated with a significantly increased risk of ROP， but also interact beyond additive and even multiplicative patterns[50]. These multiple risk factors not only contribute independently but have synergistic effects among each other. ROP has also been observed in cases without oxygen therapy in the past and this suggests that factors other than oxygen， for example， hypergly-cemia[51]， thrombocytopenia[52]， twin pregnancy[53]and assisted reproductive technology[54]may play an important role in the development of ROP. Even infants who received late retinal examination showed high incidence and prevalence of ROP， suggesting timely referral for screening of premature infants is desirable[55]. Bilirubin and/or two or more transfusions of FFP in the first week of life may help to protect preterm infants against ROP[56- 57]. Although these factors are adjustable factors， further studies are required to explore the mechanism thoroughly.

6 Conclusion

Since the pathogenesis of this disease revolves around VEGF， IGF- 1， ROS and inflammatory mediators， the potential new treatment strategies should target in regulating the level of these factors.A more profound understanding of the complex interplay of inflammatory mediators and oxidative stress is needed.The particular tendency to oxidative stress during the perinatal phase indicates that the prophylactic use of antioxidants as melatonin could help to prevent or at least reduce oxidative stress related diseases in newborns. The prevention of this disease should emphasize more around an appropriate management of blood gases derangement， infections and commonly encountered unavoidable problems such as thrombocytopenia and hyperglycemia. Maternal factors do play a role in the disease progression of ROP and this suggests that it not only commences after birth but intrauterine environment of fetus plays an immense role in disease process．

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