Contributors to programming: (1) Intrauterine environment

The critical role of intrauterine environment on fetal growth and programming has been documented in a large number of studies in humans and in animal models.Reference Whincup, Kaye and Owen^8–Reference Simmons¹⁰ Data from studies in animal models show that various nutrient and environmental manipulations during pregnancy result in fetal growth restriction, lower birth weight and programming of the offspring.Reference Whincup, Kaye and Owen^8, Reference Simmons¹⁰ Studies in humans have confirmed the original observation of Barker et al. that intrauterine growth restriction as evidenced by lower weight at birth is associated with the higher incidence of non-communicable disease in the offspring in adult life.Reference Warner and Ozanne⁹ In this context, it is interesting to note that a significantly high proportion of LBW babies have been reported to be growth retarded in utero and born small for gestational age in various studies.Reference Ehrenkranz, Younes and Lemons¹¹ However, the long-term programming consequence of the intrauterine growth restriction early in gestation cannot be easily separated from the confounding influence of other contributors to programming, particularly in studies in humans.

Contributors to programming: (2) Interruption of pregnancy

The impact of premature interruption of pregnancy on the immature baby remains undefined. The transition from fetus to neonate is accompanied by changes in respiration, circulation, glucose and energy homeostasis, thermoregulation and a host of other endocrine, metabolic and physical responses that allow for independent extra-uterine existence. Significantly, there is a marked change in the redox state and oxygenation from a relative hypoxic and markedly reduced fetal state to a highly oxygenated extra-uterine life.Reference Philippidis and Ballard^12, Reference Filippi, Messeri and Dani¹³ These changes are accompanied by generation of reactive oxygen species (ROS) and consequently changes in metabolism to compensate for the oxidant insult. ROS can cause oxidative modification of major cellular macromolecules, that is, lipids, proteins and DNA. A number of molecular targets of ROS have been identified.Reference Avery¹⁴ In the context of programming, one carbon metabolism or the methyl transfer plays a critical role in the methylation of proteins, lipids and the methylation of DNA in the transcriptional control of gene expression and metabolism.Reference Kalhan and Marczewski¹⁵ The key constituent parts of the one carbon metabolism are displayed in Fig. 1. Folate and methionine cycle form the key components of the one carbon metabolism or methyl transfers and are present ubiquitously in every cell in the body. Methyl groups from serine and glycine entering the folate cycle are transferred to homocysteine to form methionine. Methionine, an essential amino acid, is the immediate source of the methyl groups required for the methylation of proteins, nucleic acids, biogenic amines and phospholipids, etc. Methionine is converted to the bioactive compound, s-adenosyl methionine (SAM), the ubiquitous methyl donor, catalyzed by methionine adenosyl transferase. The transfer of methyl groups from SAM is catalyzed by several different methyltransferases and result in the formation of s-adenosyl homocysteine and subsequently homocysteine. Homocysteine can either be methylated back to methionine or can enter the methionine catabolic pathway, the transsulfuration cascade, wherein it condenses with serine to form cystathionine. Cystathionine, through a series of metabolic steps is converted to cysteine and ultimately to taurine. As shown in the figure, the ROS and change in redox can influence the key regulatory steps of methionine metabolism, that is, the synthesis of SAM and the transsulfuration cascade involved in the synthesis of cysteine and ultimately glutathione.Reference Prudova, Bauman and Braun^16–Reference Kabil and Banerjee¹⁸ Changes in redox and ROS could potentially affect the generation of SAM and glutathione and consequently impact the methylation process. Although not studied, perturbations in the one carbon metabolism at the time of birth could have long-lasting effect on gene expression and cause programming in the immature baby. It is important to underscore that the expression of genes regulating metabolism is an extremely orchestrated phenomenon, so that several of the metabolic processes appear at critical predetermined points during development. For example, hepatic cytosolic PEPCK and gluconeogenesis appears for the first time after birth in rodent and other mammalian species.Reference Kalhan and Parimi¹⁹ Similarly, cystathionine gamma lyase is not present in fetal liver and appears after birth.Reference Kalhan and Bier²⁰ Premature interruption of pregnancy and premature birth will result in early expression of these pathways for independent existence. The long-term consequence of these changes or programming is not known. This could be akin to modification of gene expression demonstrated in the newborn rats as a result of changes in the diet in the neonatal period.Reference Srinivasan, Laychock, Hill and Patel²¹

Fig. 1

The one carbon (methyl group) metabolism in vivo. The interrelationship between folate and methionine metabolism and its regulation are displayed. As shown, endogenous methyl groups originating from serine during its conversion to glycine are incorporated into the folate cycle to be transferred to homocysteine for the formation of methionine and ultimately s-adenosyl methionine (SAM), the universal methyl donor. The transfer of methyl groups is catalyzed by several different methyltransferases and result in the formation of s-adenosyl homocysteine (SAH) and methylated products of proteins, lipids, DNA, creatine, phosphotidyl choline, etc. SAH is then hydrolyzed to form homocysteine. Homocysteine is the branch point of the methionine cycle, is either methylated back to methionine or participates in the transsulfuration pathway whereby the sulfhydryl group is transferred to serine to form cysteine. Cysteine is a component of the tripeptide, glutathione, the key intracellular antioxidant. The one carbon metabolism requires folate, vitamin B₁₂ (B₁₂) and vitamin B₆ (B₆). Homocysteine can lead to protein modification, induce inflammation, endothelial damage, thrombosis, etc. The formation of SAM from methionine (catalyzed by methionine adenosyl transferase) is sensitive to reactive oxygen injury (ROS).Reference Prudova, Bauman and Braun^16–Reference Kabil and Banerjee¹⁸ Transsulfuration is regulated by change in redox state, as well as by hormones such as insulin and glucagon. Thus at the time of birth, both transmethylation and transsulfuration can be effected by the rapid change in redox, by the increased generation of ROS and by the birth associated acute surges in several hormones. GNMT, glycine n-methyltransferase. THF, Tetrahydrofolate; GSH, glutathione; MS, methionine synthase; CbS, cystathionine beta synthase; CgL, cystathionine gamma lyase.

Contributors to programming: (3) Clinical care protocols in the NICU

Evaluation of the contribution of the clinical protocols, used in the NICU, to programming is confounded by a number of variables, making it difficult to establish cause and effect relationship. The potential variables include:

1. (1) Heterogeneity of the infant population. As shown in Table 1, infants admitted to the NICU represent a wide range of gestational age and therefore development. These data also show that, over the last 20 years, the relative proportion of various gestation groups and their survival rate has not changed significantly. Since the potential for vulnerability to environmental and other insults may be different at different stages of development, these data underscore the need to examine these gestation groups separately when examining the possible programming effects.

2. (2) The clinical intervention for cardio-respiratory support, for treatment of sepsis, fluid therapy and other problems related to prematurity such as hyperbilirubinemia, necrotizing enterocolitis, patent ductus arteriosus, hypoglycemia and anemia, could have added modifying influences. In addition, these interventions can be institution specific depending on the local expertise and bias and are not easily quantifiable in data analysis.

3. (3) The protocols for clinical care have continuously evolved over time by incorporating new innovations including pharmacological, clinical, nutrition and other technologies. In addition, certain pharmacological interventions, considered to be useful early on, have been discontinued as the potential harmful effects were recognized. This has been specifically true for the postnatal use of glucocorticoids (dexamethasone) for the prevention and treatment of bronchopulmonary dysplasia after reports of neurodevelopmental impairment were published.

4. (4) The algorithms of clinical care have other variations introduced by individual care providers based upon the clinical condition of the baby, or the result of individual bias and expertise resulting in the achieved goal being quite different from that planned or desired. These changes in the clinical protocols make it difficult to correlate the observations in the physiological phenotype in adult with the neonatal intervention.

The commonly used clinical interventions in the NICU that could have programming effects on the baby are listed in Table 2. For most of these, the actual cause and effect has not been demonstrated. In the following, we will discuss some of the scientific evidence for potential programming and epigenetic effects of these interventions. Additionally, the LBW babies are exposed to environmental factors such as noise (from the isolette and other equipment), light and other pharmacological interventions like caffeine, oxygen, etc. Their effects, if any, in relation to programming have not been determined.

Table 2

Clinical interventions that may have programming effects

Nutritional care in the NICU

Nutrition has been recognized as the major or critical influence in programming during development. Nutrient imbalance at critical periods during development has been suggested to result in long-term programming by causing epigenetic changes in the DNA and by modifications of the histones.Reference Symonds, Sebert, Hyatt and Budge²² Over the past many years the protocols and algorithms for the nutritional management of the LBW baby have been evolving continuously with the goal of providing enough energy producing substrates and proteins in order to achieve a rate of accretion of lean body mass and body weight that resembles the rate of growth and protein accretion of fetus in utero. Whether such a goal is sufficient for optimal growth and development, or is it desirable or can it be achieved has been a subject of clinical debate and scientific inquiry for many years and continues to be discussed in the contemporary literature.Reference Hay and Thureen²³ As discussed below these nutritional protocols have resulted in varying rates of growth of the babies in the neonatal period and possible long-term consequences.

Parenteral and enteral protein intake

The quality and quantity of protein, the route (parenteral v. enteral) of administration and the duration of parenteral nutrition have seen the most dramatic change in the last 20–30 years. With the increasing emphasis on early introduction of enteral feeding, due to its recognized clinical benefits, the average duration of parenteral nutrition has decreased remarkably from an average of ~30 to 40 days to the current duration of ~10 days in most NICUs.Reference Ehrenkranz, Younes and Lemons¹¹ In addition, as the protein requirements of the LBW babies were recognized and the potential impact of low protein intake were documented, there has been a greater emphasis on early provision of larger amounts of parenteral amino acids to the LBW babiesReference Hay and Thureen^23, Reference Hay and Thureen^26–Reference Ibrahim, Jeroudi, Baier, Dhanireddy and Krouskop³⁰ such that a higher proportion of LBW babies are now given 3–4 g/kg day amino acids starting on day one after birth, with no clinically measurable adverse consequences.Reference te Braake, van den Akker, Wattimena, Huijmans and van Goudoever^28–Reference Ibrahim, Jeroudi, Baier, Dhanireddy and Krouskop³⁰ This is in contrast to the clinical practice of 20 and 30 years ago when these infants routinely did not receive amino acids in their parenteral nutrient solutions on the first 2–3 days after birth or received very low quantities (0.5–1.0 g/kg day) often accompanied by low caloric intake in the form of glucose alone.

Low protein intake

The impact of low protein intake on long-term programming in humans has not been reported in part because the follow-up period has been relatively short and because of the confounding effect of other clinical and biological variables on this population. In this context, it is important to underscore the differential effect of low protein with low energy intake as compared with low protein intake in the presence of isocaloric energy intake. Although the combination of low energy intake along with low protein intake results in metabolic responses akin to starvation, that is, increased rate of lipolysis from the adipose tissue and an increased rate of whole body proteolysis in order to meet the energy requirements, the isocaloric protein deficiency results in markedly different responses. In the rodent, dietary protein restriction during pregnancy results in fetal growth retardation and metabolic programming resulting in hypertension, impaired beta cell function and mass, impaired insulin sensitivity and other pathological responses in the adult offspring.Reference Warner and Ozanne⁹ These have been associated with a change in the hypothalamic–pituitary–adrenal axis, changes in renin–angiotensin system and alterations in the concentrations of catecholamines and adrenoreceptors.Reference Warner and Ozanne^9, Reference Augustyniak, Singh, Zeldes, Singh and Rossi³¹ The perturbations in the maternal metabolism that lead to the observed programming responses in the fetus have not been delineated. In the non-pregnant adult rat, isocaloric protein restriction for 7–10 days resulted in significant differential expression of a number of genes involved in cell cycle, cell differentiation, transport, transcription and metabolic processes in the liver.Reference Kalhan, Uppal and Moorman³² The expression of genes involved in fatty acid oxidation and serine biosynthesis was higher while those involved in urea synthesis and fatty acid synthesis was lower in the protein restricted animals. Tracer isotope studies showed that protein restriction caused a 50% increase in de novo synthesis of serine. Since serine is the primary source of methyl groups for transmethylation in vivo, it was interesting to note that the measured rates of transmethylation of methionine and the methylation potential [SAM/s-adenosyl homocysteine (SAH) ratio] was significantly increased in the protein restricted animals. These data were interpreted to suggest a high methylation demand placed on the organism as a result of changes in cell cycle and differentiation. Chronic protein malnutrition in humans identified by low plasma transthyretrin levels was observed to be associated with hyperhomocysteinemia and possibly changes in one carbon metabolism.Reference Ingenbleek, Hardillier and Jung³³ Whether the LBW baby responds to dietary protein restriction in a similar manner has not been evaluated. In addition, the long-term consequences of such a response or its programming effects, if any, have not been determined. It can only be speculated that isocaloric protein restriction at this critical period of development, potentially, can cause programming effect by changes in the one carbon metabolism, transmethylation and methylation potential. However, the clinical data from earlier studies in the LBW babies do not show evidence of isolated low protein intake but show both low protein and energy intake during the first few days after birth.Reference Olsen, Richardson, Schmid, Ausman and Dwyer^24, Reference Embleton, Pang and Cooke³⁴ The early protein energy malnutrition in the NICU has been related to neurodevelomental deficits at age of 18 months.Reference Stephens, Walden and Gargus³⁵ Perhaps these developmental deficits also should be considered programming affects.

High protein intake

As discussed above, there has been a recent concerted effort to promote higher protein intake by both enteral and parenteral route and to initiate enteral feedings as early as possible. The higher intake of protein via the enteral route has not been reported to cause any significant problems. In contrast, intravenously administered amino acids mixtures because of their unique composition can cause immediate metabolic changes which may have programming affects. The common amino acid mixture used in the NICU in United States was formulated to result in plasma amino acid concentration resembling that of a term to 1-month-old breastfed infant and does not resemble the amino acid composition of any protein.Reference Heird, Hay and Helms³⁶ The additional criteria for the formulation were maintenance of solubility and stability of the mixture. In general the commonly used amino acid mixtures (e.g. Trophamine) are high in branched chain amino acids and in methionine and low in cysteine. The early administration of larger (3 g/kg day) amounts of amino acids results in higher plasma concentrations of all amino acids in particular essential amino acids for a variable period without causing any toxic effects.Reference Blanco, Gong and Green³⁷ In addition, aggressive early administration of higher amount of amino acids did result in positive nitrogen balance and growth of the neonate.Reference te Braake, van den Akker, Wattimena, Huijmans and van Goudoever^28, Reference Ibrahim, Jeroudi, Baier, Dhanireddy and Krouskop³⁰ Studies done by Battista et al.Reference Battista, Price and Kalhan³⁸ showed that the majority of excess branched chain amino acids (BCAA) in Trophamine are oxidized to CO₂ and urea and are not retained in body proteins (Fig. 2). Similarly, essential amino acid methionine, also administered in larger quantities than its estimated daily requirement (~28 μmol/kg h as compared with the estimated requirement of 16 μmol/kg h) is also transsulfurated and oxidized to CO₂.Reference Thomas, Gruca and Bennett³⁹ However, for it to enter transsulfuration pathway, the excess methionine enters the transmethylation cycle, and is converted to SAM, SAH and homocysteine (Fig. 1). The higher transmethylation will result in an excess methyl load on the organism. Although the consequences of the excess methyl load on the LBW baby are not known, data from animal models show that excess methyl load during pregnancy in mice can lead to the development of allergic asthma in the offspring.Reference Hollingsworth, Maruoka and Boon⁴⁰ Corresponding data in humans have been inconsistent.Reference Haberg, London, Stigum, Nafstad and Nystad^41, Reference Whitrow, Moore, Rumbold and Davies⁴² However, it is significant to note that bronchial asthma has consistently been identified as a significant morbidity at follow-up in LBW babies in several studies.Reference Hack^3, Reference Doyle and Anderson⁵ The contribution of excess methyl load in the parenteral nutrition on long-term programming requires careful longitudinal follow-up studies.

Fig. 2

Metabolism of leucine and methionine in preterm infants receiving parenteral amino acid nutrition (upper panel). The effect of branched amino acid enriched parenteral nutrition (Trophamine) on rates of leucine oxidation and urea synthesis in preterm babies. The rates of leucine oxidation were measured using [1-¹³C]leucine tracer and the rate of synthesis of urea using [¹⁵N₂]urea tracer. As shown, excess BCAA were oxidized to CO₂ and urea (lower panel). Rate of appearance of methionine (Ra) in the plasma and estimates of transmethylation and transsulfuration in healthy full-term babies and preterm babies receiving parenteral amino acid nutrition. As shown, the parenteral amino acid containing higher than required methionine (high Ra) resulted in increased transmethylation and transsulfuration. BCAA, branched chain amino acids.

Growth from ‘Birth to Term’

Data reported by a number of investigators from clinical centers across the world have underscored the difficulties in achieving the goal of growth in the neonatal period that will mimic the rate of intrauterine growth. These studies show that in spite of our best efforts in clinical and nutritional care of the LBW baby, these babies at the time of discharge from the hospital do not achieve the median birth weight of the reference fetus at the same postmenstrual age.Reference Ehrenkranz, Younes and Lemons^11, Reference Olsen, Richardson, Schmid, Ausman and Dwyer^24, Reference Cooke, Ainsworth and Fenton²⁵ Although early and aggressive parenteral and enteral nutritional strategies have been shown to attenuate this postnatal growth failure, the goal of achieving intrauterine rate of growth remains allusive.Reference Cooke, Ainsworth and Fenton^25, Reference Embleton, Pang and Cooke^34, Reference Wilson, Cairns and Halliday^43, Reference Dinerstein, Nieto and Solana⁴⁴ The postnatal growth retardation or the so called ‘birth to term’ growth failure has been described as an inevitable and universal problem in preterm infants.Reference Embleton, Pang and Cooke³⁴ The potential contributors to the birth to term growth failure, in addition to nutritional support, include neonatal morbidities such as lung disease, necrotizing enterocolitis, intraventricular hemorrhage, treatment with glucocorticoids, etc. Statistical model analysis of the various contributors to the postnatal growth showed that medical morbidities and nutritional factors, in particular protein intake, explained ~50% of the variance in the overall growth velocity.Reference Olsen, Richardson, Schmid, Ausman and Dwyer²⁴ Body composition studies of these infants using dual emission X-ray absorptiometry (DEXA) showed reduced linear growth and reduced fat free mass coupled with increased global and central fat mass.Reference Cooke and Griffin⁴⁵ Whether such body composition changes at this early stage in life have any relation to the development of insulin resistance and metabolic syndrome later in life remains to be examined. Comparison with the studies from India of ‘fat thin baby’ would suggest a high risk of development of insulin resistance later in life.Reference Yajnik, Fall and Coyaji⁴⁶ A significant relationship between growth velocity in the NICU and the likelihood of neurodevelopmental impairment has also been reported.Reference Stephens, Walden and Gargus^35, Reference Ehrenkranz, Dusick and Vohr⁴⁷ Whether the impaired neurodevelopment is the consequence of programming or due to an acute irreversible injury to the brain is not clear.

Pharmacological interventions

A number of pharmacological agents, commonly used in the NICU, are listed in Table 2. Data on their long-term consequence are limited. Some of these agents such as caffeine, surfactant are administered to almost all LBW babies and therefore their consequences are difficult to delineate. Others like nitric oxide (NO), erythropoietin, VEGF (vascular endothelial growth factor) inhibitors have only recently been introduced in clinical care and therefore no long-term follow-up data are available as yet. In the following, available data on some of these are discussed in the context of their potential role in programming.

Vitamin A

The plasma concentrations of retinol and retinol binding protein levels in LBW babies have been observed to be low in a number of studies. These low levels do not appear to be influenced by the amount and route of feeding and remain low for several months after discharge from the hospital.Reference Peeples, Carlson, Werkman and Cooke^48–Reference Shenai, Chytil and Stahlman⁵¹ These data have been interpreted to represent a state of vitamin A deficiency. Data from studies in vivo and in vitro have demonstrated a critical role of vitamin A in the differentiation of pulmonary cells, in surfactant protein metabolism, in airway growth and expression of several genes involved in the development of lungs. In addition, clinical observation studies had suggested that poor vitamin A status in the first months after birth in LBW babies was associated with an increased risk of developing bronchopulmonary dysplasia and long-term respiratory disability.Reference Shenai, Chytil and Stahlman^51, Reference Spears, Cheney and Zerzan⁵² Based upon these data, clinical trials of oral or parenteral vitamin A supplement have been done. These studies show that administration of vitamin A during the first 4 weeks after birth results in significant but small reduction in death or oxygen use at 36 weeks postmenstrual age.Reference Shenai, Kennedy, Chytil and Stahlman^53–Reference Tyson, Wright and Oh⁵⁶ Even though the clinical impact of vitamin A supplement was small, it was recommended that 5000 IU of vitamin A be given intramuscularly to LBW babies who are at risk for developing chronic lung disease or bronchopulmonary dysplasia. However, such a recommendation is not uniformly followed by the clinical neonatologists.Reference Darlow and Graham^57, Reference Barros, Bhutta and Batra⁵⁸

The long-term consequences and potentially programming effects of the administration of vitamin A to the LBW infants have not been delineated. Limited clinical data of relatively short follow-up period suggests that there are no identifiable acute toxicity of high dose vitamin A in the neonate. In addition, no significant impact on mortality and neurodevelopmental impairment at 18–22 months of age was observed.Reference Tyson, Wright and Oh^56, Reference Ambalavanan, Tyson and Kennedy⁵⁹ However, high dose vitamin A could potentially have programming effect by modulating SAM dependent transmethylation reactions (Fig. 1). Studies in rat show that high dose vitamin A and its derivatives (all-trans retinoic acid) caused an increase in abundance and activity of glycine n-methyltransferase (GNMT) and hypomethylation of DNA in the liver.Reference Rowling, McMullen and Schalinske^60–Reference Rowling and Schalinske⁶³ Interestingly, the observed responses were both tissue and gender specific, so that there was no effect of retinoids on pancreatic and renal GNMT activity.Reference McMullen, Rowling, Ozias and Schalinske⁶² In addition, the increase in hepatic GNMT activity was less in female rats when compared with the males. The long-term phenotypic effect of these changes in methylation has not been reported. However, exposure of rats to very high (80,000 IU/kg day) doses of vitamin A during 1–5 days after birth has been shown to result in long-lasting (at 36 weeks of age) defect in learning in water-maze task and depression in heat–pain responses.Reference Kihara, Matsuo, Sakamoto, Yasuda and Tanimura⁶⁴ Other data suggestive of programming effect on immune system have been reported in response to neonatal exposure to vitamin A in rats.Reference Csaba, Kovacs and Pallinger⁶⁵ A pattern of motor deficit following late embryonic exposure to all-trans retinoic acid has also been reported.Reference Coluccia, Borracci, Belfiore, Renna and Carratu⁶⁶ Future studies will examine whether these observations are due to a programming effect related to changes in methylation status caused by perturbation in methionine cycle.

Glucocorticoids

Glucocorticoids have been and remain the most extensively used pharmacological intervention used in the perinatal period. Data from animal models have clearly demonstrated the programing effect of glucocorticoids administration during development. These studies show that exposure to excess glucocorticoids in the prenatal period either due to increased endogenous production as a result of maternal stress, or by exogenous administration or as a result of inhibition of 11-β hydroxysteroid dehydrogenase type 2 (the placental barrier to maternal glucocorticoids) results in lower birth weight and causes hyperglycemia, hypertension, increased HPA axis activity and behavior abnormalities in the offspring.Reference Harris and Seckl^67, Reference Drake, Tang and Nyirenda⁶⁸ These effects have been observed to be transmitted across generations suggesting an epigenetic mechanism. It should be underscored that while animal models are critical for our understanding of the biological and molecular mechanisms of programming, the doses of pharmacological agents used and the duration of administration in these studies have been quite different than those used in clinical practice. In clinical practice, glucocorticoids are either used antenatally to induce pulmonary surfactant synthesis in case of impending premature delivery or in the neonate with respiratory distress syndrome or bronchopulmonary dysplasia in order to wean them from respiratory/ventilator support and minimize the risk of lung injury.

Nitric Oxide (NO)

NO, a major endogenous regulator of vascular tone (endothelial vascular relaxing factor) and intracellular signaling molecule, is being used extensively for the treatment of pulmonary hypertension in the neonates born at term or at near-term gestation.Reference Flier and Barrington⁸¹ Systematic reviews and meta-analysis of the published literature have confirmed the effectiveness of inhaled NO in reducing death and the need for ECMO (extracorporeal membrane oxygenation) without any measurable systemic toxicity in the doses employed.Reference Flier and Barrington⁸¹ Although not approved for use in the prematurely born babies, it is often used outside the licensed indication (off-label) in preterm babies for the prevention and treatment of bronchopulmonary dysplasia. The rational for the use of inhaled NO in the prematurely born infants, and its clinical effectiveness (or lack of it) have been discussed.Reference Subhedar and Dewhurst^82–Reference Cole, Alleyene and Barks⁸⁴ The potential acute toxic effects of inhaled NO include acute lung injury by peroxynitrite formed by the reaction of NO with oxygen, impaired platelet aggregation, methemoglobinemia and possibly extrapulmonary vasodilatation.Reference Weinberger, Laskin, Heck and Laskin⁸⁵ NO is rapidly inactivated by hemoglobin to form methemoglobin. The extrapulmonary effects of inhaled NO have been attributed to its binding to circulating albumin or hemoglobin and thus delivery of NO in its active form to systemic circulation.Reference Weinberger, Laskin, Heck and Laskin⁸⁵ In addition, NO is also considered to be an epigenetic molecule.Reference Illi, Colussi and Grasselli⁸⁶ NO exerts its effect on gene expression and regulation via nitrosylation and tyrosine (tyr)-nitration of a number of nuclear and non-nuclear proteins.Reference Illi, Colussi and Grasselli⁸⁶ The epigenetic effects of NO have particularly been studied in the developing neurons.Reference Nott and Riccio⁸⁷ Although not examined in relation to therapeutic doses of NO used in the premature infants, the transfer of NO as protein bound adductReference Keanney, Simon and Stamler⁸⁸ could potentially allow for its transport to distant organs and consequently result in epigenetic modification of gene expression. Carefully conducted studies in vivo and in vitro would delineate such effects in the future.

Anti-VEGF therapy

Bevacizumab (Avastin), an anti-VEGF therapy, previously used to treat diabetic retinopathy and macular degeneration in adults is now being implemented in preterm NICU infants between 30 and 40 weeks post menstrual age for the treatment of retinopathy of prematurity. It is a recombinant humanized vascular endothelial VEGF antibody that prevents VEGF from binding to its receptors.Reference Lin, Nguyen and Mendoza⁸⁹ Bevacizumab binds to all isoforms of VEGF,Reference Ferrara⁹⁰ blocks VEGF-induced angiogenesis and is approved by the US Food and Drug Administration for intravenous treatment of metastatic colon cancer. A recent randomized controlled trial of intravitreal injection of bevacizumab for stage3+ retinopathy of prematurity in the United States has shown lower rates of recurrence of retinopathy (anti-VEGF: 6% v. laser: 42%), rates of macular dragging (3% v. 48%) and retinal detachment (0% v. 6%) in the bevacizumab group compared with the laser therapy group.Reference Mintz-Hittner, Kennedy and Chuang⁹¹ Although seemingly an exciting new therapy with great potential to minimize serious sequelae of prematurity, information on the long-term implications of this therapy remains sparse. Intravitreal bevacizumab enters the general circulation, results in prolonged VEGF inhibition and has a half-life of up to 2 weeks in primates. In the fetus, VEGF is expressed in most tissues and is critical for growth and development of vital organs such as kidneys, lungs and brain during the third trimester. In-vitro studies on the effect of bevacizumab-induced inhibition of VEGF have demonstrated dose-dependent inhibition of human umbilical vein endothelial cell proliferation,Reference Wang, Fei, Venderlaan and Song⁹² increased mortality and impaired liver and cartilage growth in newborn miceReference Gerber, Hillan and Ryan^93, Reference Gerber, Vu and Ryan⁹⁴ and measurable levels of anti-VEGF drug in kidneys, spleen, lung, opposite eye and brain of newborn rabbits receiving intravitreal injection in a single eye.Reference Wu, Lai and Chen⁹⁵ Preterm infants with retinopathy of prematurity (ROP) have a compromised blood–retinal barrier that may allow even more bevacizumab to enter the blood stream. Given the fact that preterm infants receive treatment for ROP at a time when growth and differentiation is maximal, the potential of such a therapy on programming requires careful evaluation.

Others

Bisphenol-A (BPA), an estrogen-mimic endocrine disrupter, is extensively used in manufacturing polycarbonate plastic and epoxy resins from which food and beverage containers and dental materials are made.Reference Maffini, Rubin, Sonnenschein and Sotoi^96, Reference Calafat, Weuve and Ye⁹⁷ BPA may also be used in polyvinyl chloride (PVC) industry. PVC is used in the manufacture of several medical products used in the NICU such as feeding tubes, umbilical vessel catheters, bags and tubings for parenteral fluids and for their administration, endotracheal tubes, respiratory masks, etc. Di(2-ethylhexyl) phthalate is a plasticizer added to PVC in a number of these medical products to make them soft and pliable. Calafat et al. Reference Calafat, Weuve and Ye⁹⁷ in a small study examined the exposure to BPA and other phenols in premature infants in two NICUs in Massachusetts, USA. Their data showed that the geometric mean BPA concentration among premature infants undergoing intensive medical interventions was one order of magnitude higher than that seen in the general population. In addition, they found that the conjugated species of phenols were the primary urinary metabolites suggesting that the premature LBW babies had some capacity to metabolize BPA. Whether such an exposure to BPA in the LBW babies has any programming effects continues to be an important area of investigation. Data from studies in animal models show that exposure to BPA during early development can change the phenotype of the offspring by stably altering the epigenome.Reference Dolibnoy, Huang and Jirtle⁹⁸ Interestingly, administration of methyl donors such as folic acid negated the DNA hypomethylating effect of BPA in this study. Other studies have shown disruptive effects of BPA on the expression of sexually selected traits in adults as a consequence of developmental exposure in the mouse and alterations in the development of mammary gland in the rhesus monkey.Reference Jasarevic, Sieli and Twellman⁹⁹

Contributors to programming: (4) Catch-up and catch-down growth during infancy

With the improvement in nutritional care of the preterm babies after discharge from the hospital, the frequency of growth failure or stunting has markedly decreased and most prematurely born LBW babies show evidence of catch-up growth.Reference Hack, Merkatz, McGrath, Jones and Fanaroff^100–Reference Itabashi, Mishina and Tada¹⁰² However, weight gain alone should not be considered evidence of catch-up growth. Uthaya et al. studied the body composition of preterm LBW babies (<32 weeks gestation) using whole body MRI technique.Reference Uthaya, Thomas and Hamilton¹⁰³ Their data show that at the postconceptional age of term gestation, the preterm babies had significant decrease in subcutaneous adipose tissue and significant increase in visceral adipose tissue while accelerated weight gain in these babies was accompanied by increased total and subcutaneous adiposity. They suggested that preterm baby may be at risk in later life of metabolic complications through increased and aberrant adiposity. However, a study by Cooke et al.Reference Cooke, Griffin and McCormick¹⁰⁴ of preterm infants fed a nutrient enriched formula did not find evidence of altered adiposity measured longitudinally during first 6 months using DEXA method. DEXA does not differentiate subcutaneous from visceral fat. A number of other studies, reviews and systematic analysis have examined the impact of after hospital discharge nutrient management of LBW babies and catch-up and catch-down growth.Reference Yeung^105–Reference Monteiro and Victora¹⁰⁷ In the majority of the studies LBW does not represent the LBW graduate of the NICU, but rather small for gestational age full-term babies.Reference Yeung^105–Reference Monteiro and Victora¹⁰⁷ Outcome measurements in these infants are confounded by their response to the adverse intrauterine environment. Additionally, catch-up growth in the LBW preterm babies has been observed to continue until 6 years of age.Reference Wehkalampi, Hovi and Dunkel¹⁰⁸ Accelerated growth or rapid weight gain has some distinct clinical advantages related to decrease in infections and improved rate of survival.Reference Yeung¹⁰⁵ However, it also has been consistently related to the development of obesity in later life in both appropriate for gestational age and small for gestational age full term and preterm infants.Reference Bazaes, Alegría and Pittaluga^109, Reference Stettler and Iotova¹¹⁰ A detailed analysis of these data are beyond the scope of this review and the reader is referred to some excellent publications on the subject. Obesity, in particular visceral adiposity, is associated with the insulin resistance and metabolic syndrome.

Clinical outcomes and their correlates

Follow-up data show that the LBW babies have a higher incidence of hypertension, visceral obesity, asthma and neurodevelopmental and behavior problems and perturbations in glucose insulin homeostasis. Whether these non-communicable disorders are the consequence of programming as a result of events during development has been examined only in the case of insulin resistance. These analyses are by necessity statistical correlations of observational data and do not represent causality. The reported data on insulin resistance are discussed below.