Neonatal cerebral white matter injury in preterm infants is associated with culture positive infections and only rarely with metabolic acidosis

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1 American Journal of Obstetrics and Gynecology (2004) 191, 1305e10 Neonatal cerebral white matter injury in preterm infants is associated with culture positive infections and only rarely with metabolic acidosis Ernest M. Graham, MD, a Cynthia J. Holcroft, MD, a Karishma K. Rai, BA, b Pamela K. Donohue, ScD, c Marilee C. Allen, MD c Department of Gynecology and Obstetrics, Division of Maternal-Fetal Medicine, Johns Hopkins University School of Medicine, Baltimore, Md, a Ross Medical School, Dominica, West Indies, b and Department of Pediatrics, Division of Neonatology, Johns Hopkins University School of Medicine, Baltimore, Md c Received for publication February 2, 2004; revised May 22, 2004; accepted June 11, 2004 KEY WORDS Periventricular leukomalacia Neonatal metabolic acidosis Neonatal infection Objective: Neonatal cerebral white matter injury represents a major precursor for neurological impairment and cerebral palsy. Our objective was to identify risk factors associated with its development. Study design: This retrospective case-control study of all births between 23 and 34 weeks gestation at a single university hospital between May 1994 and September 2001 identified 150 cases with white matter injury characterized by periventricular leukomalacia or ventricular dilatation from white matter atrophy that were chromosomally normal and did not have other congenital anomalies. Cases were matched to controls without brain injury by the next delivery within 7 days of their gestational age. Results: There were small differences between controls and cases in gestational age (27.5 G 2.7, 27.4 G 2.6 weeks, P =.01) and birth weight (1053 G 402, 966 G 285 g, P =.002) that were statistically but not clinically significant. There was no difference in the percentage of controls and cases delivered by cesarean (45%, 49%, P =.64). There were no differences between controls and cases in umbilical arterial ph (7.27 G 0.11, 7.25 G 0.15, P =.19), base excess (e2.1 G 2.7, e3.0 G 4.1 mmol/l, P =.28), ph less than 7.0 (2/122 [2%], 3/107 [3%], P = 1.0), or base excess less than e12 mmol/l (4/121 [3%], 6/106 [6%], P =.75). The cases had a significant increase in positive blood (19%, 29%, P =.036), cerebrospinal fluid (6%, 17%, P =.002), and tracheal (9%, 22%, P =.003) cultures during the neonatal period. Conditional logistic regression showed a significant association among multiple gestations (P =.02), intraventricular hemorrhage (P!.001), and positive tracheal cultures (P =.02) with cerebral white matter injury. Conclusion: Culture-positive infection was associated with an increased risk of cerebral white matter injury in preterm neonates. Intrapartum hypoxia-ischemia as manifested by metabolic acidosis was rarely associated with white matter injury and was not different from the incidence in premature neonates without injury. Ó 2004 Elsevier Inc. All rights reserved. Presented at the Twenty-Fourth Annual Meeting of the Society for Maternal-Fetal Medicine, February 2-7, 2004, New Orleans, La. Reprints not available from the authors /$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi: /j.ajog

2 1306 Graham et al Neonatal cerebral white matter injury, characterized by periventricular leukomalacia (PVL) and ventricular dilatation, represents a major precursor for neurological impairment and cerebral palsy in later life. PVL is the most common form of brain injury in premature infants and results in cerebral palsy in 60%-100% of survivors. 1 Cerebral white matter is extremely sensitive to perfusion-related injury because it receives only 25% of the blood flow of cortical gray matter 2 and has a maturation-dependent impairment of the ability to autoregulate blood flow. Blood flow to the cerebral white matter occurs through short penetrating and long penetrating arteries. 3 The short penetrators extend only into the subcortical white matter, and the long penetrating arteries terminate in the deep periventricular white matter. The border zones between these arteries are most susceptible to a fall in perfusion pressure and cerebral blood flow. As the fetus matures, there are increases in the number of short penetrators and anastomoses between the short and long penetrators, leading to a decrease in vulnerable end zones in the subcortical and central cerebral white matter so that white matter injury becomes less frequent. Although cerebral white matter injury is most common in premature infants, it can occur in term infants subjected to hypoxic-ischemic insults. PVL refers to necrosis of white matter in a characteristic distribution dorsal and lateral to the external angles of the lateral ventricles and less severe injury to the white matter peripheral to these areas of focal necrosis. The principal findings on ultrasound are cystic lesions and ventriculomegaly with diminished cerebral white matter volume. Bilateral spastic cerebral palsy is leg dominated in most cases, which is due to the fact that the pyramidal projections to the legs are located closer to the ventricular wall than projections to the arms and so are more easily affected by white matter pathologies that extend from the ventricles in a lateral direction. It has been shown in adolescents born prematurely with PVL that dysfunction of the arms was observed only in patients with a lateral lesion extent of 23 mm or greater and that leg dysfunction was already present in patients with a lateral lesion extent of 20 mm. 4 The objective of this study was to determine factors associated with cerebral white matter injury and, because this area of the brain is extremely sensitive to a decrease in blood flow, to examine the role of hypoxia-ischemia as manifested by metabolic acidosis in this neonatal brain pathology. Material and methods This is a retrospective case-control study of all births between 23 and 34 weeks gestation at a single tertiary university hospital between May 1994 and September During this 7-year, 4-month period, 150 cases with white matter injury characterized by PVL or ventricular dilatation because of white matter atrophy that were chromosomally normal and did not have other anomalies were identified. Each of these 150 cases was matched to 150 controls by using the next delivery within 7 days of the same gestational age. All infants born before 32 weeks had at least 3 head ultrasounds. Infants born between 32 and 34 weeks had a head ultrasound if it was felt warranted by the attending neonatologist but not on a routine basis. For all infants born before 32 weeks, the first head ultrasound was done hours after birth, and a second ultrasound was done at days of life. If intracranial bleeding was seen on the second ultrasound, a follow-up ultrasound was done in 1-2 weeks to look for progressive ventricular dilatation. All infants born before 32 weeks had a head ultrasound at 6 weeks of life to specifically look for PVL. Maternal and neonatal records were reviewed. It is the policy of our institution to obtain an umbilical arterial gas on all deliveries; however, in our study an umbilical arterial gas was obtained in 122 of 150 (81%) of controls and 107 of 150 (71%) of cases. We were not able to obtain a cord gas in 100% of deliveries because obtaining an adequate amount of arterial blood is much more difficult in preterm deliveries in whom the vessels are much smaller. The clinical diagnosis of chorioamnionitis was made in the presence of maternal fever, with the presence of at least one other finding of fetal tachycardia, uterine tenderness, or purulent vaginal discharge. Patients diagnosed with clinical chorioamnionitis were immediately started on intravenous ampicillin and gentamicin if not allergic. All the placentas in the study were examined by an attending pathologist at our institution. Histologic chorioamnionitis was diagnosed when any polymorphonuclear leukocytes were seen in either the chorion or amnion or in significant amounts in the subchorionic space. Histologic funisitis was diagnosed when polymorphonuclear leukocytes were seen in the umbilical cord. Continuous data were compared using a paired t test, and categoric data were compared using McNemar chisquare with P!.05 being considered significant. Conditional (fixed effects) logistic regression was used to determine which variables might be associated with the presence of neonatal cerebral white matter brain injury. All variables with P!.10 in the univariate analysis were included in the conditional logistic regression model. Analysis was performed using Stata 7.0 (Stata Corporation, College Station, Tex). Results When maternal demographics were compared for the cases and controls, there was no difference found in maternal age, gravity, parity, or race (Table I). Each

3 Graham et al 1307 Table I Maternal demographics Controls (n = 150) Cases (n = 150) P value OR, 95% CI Age (y) 25.7 G G Gravity (median) Parity (median) Race.10 White 41 (27%) 58 (39%) Black 106 (71%) 86 (57%) Other 3 (2%) 6 (4%) Gestational age (wk) 27.5 G G * Birth weight (g) 1053 G G * Cesarean 68 (45%) 73 (49%) , IUGR 14 (9%) 18 (12%) , Oligohydramnios 9 (6%) 14 (9%) , Multiple gestation 21 (14%) 36 (24%).017* 2.50, Steroids 120 (80%) 119 (79%) , Magnesium 93 (62%) 98 (65%) , Meconium 6 (4%) 8 (5.3%) , IUGR, Intrauterine growth restriction; OR, odds ratio; CI, confidence interval. Table II Reason for preterm delivery Controls Cases P value OR, 95% CI Premature 100 (67%) 98 (65%) , labor PPROM 66 (44%) 54 (36%) , Preeclampsia 23 (15%) 23 (15%) , Abruption 12 (8%) 13 (9%) , Previa 4 (3%) 2 (1%) , PPROM, Preterm premature rupture of membranes; OR, odds ratio; CI, confidence interval. Table III Neonatal complications Controls Cases P value OR, 95% CI RDS 106 (71%) 100 (67%) , CLD 58 (39%) 90 (60%)!.001* 3.29, NEC 21 (14%) 24 (16%) , Sepsis 80 (53%) 88 (59%) , Death 23 (15.3%) 6 (4%).002* 4.40, IVH 31 (21%) 82 (55%)!.001* 4.92, Seizures 6 (4%) 4 (3%) , RDS, Respiratory distress syndrome, CLD, chronic lung disease, NEC, necrotizing enterocolitis; IVH, intraventricular hemorrhage; OR, odds ratio; CI, confidence interval. control was matched to the preceding case by gestational age within 7 days, and even though the mean and standard deviation are almost identical for the cases and controls, paired t test analysis showed a statistically significant difference in gestational age in the matched pairs. Although the incidence of white matter injury is closely linked to gestational age because all cases have white matter injury and all controls are without brain injury, this small difference in gestational age is not clinically significant and should not have any effect on umbilical arterial blood gas values or the incidence of neonatal infection. There is a small 87-g difference in birth weight between cases and controls that is statistically but not clinically significant. There was a significantly increased incidence of multiple gestations among the cases with cerebral white matter injury. For all multiple gestations, 4 of 21 (19.0%) of the controls and 6 of 36 (16.7%) of the cases had a monochorionic placenta. The incidence of cesarean delivery between cases and controls was similar. The incidence of antenatal steroid and magnesium exposure, both of which may potentially affect neurologic morbidity, were not significantly different between the groups. The reasons for preterm delivery were similar between cases and controls as shown in Table II, with preterm labor and preterm premature rupture of membranes being by far the most common. Another potential confounder, preeclampsia, which has been shown to be strongly protective against cerebral palsy in preterm infants, 5 was the same in both groups. The incidence of neonatal complications was examined between the cases and controls (Table III). There was a significantly higher incidence of neonatal death in the controls. Many of the controls may have died before they could have possibly developed cerebral white matter injury. The lower incidence of death among the cases is as expected because the neonate would have to live for some time, probably around 6 weeks, to develop ultrasonographic features of white matter injury. The cases had a higher incidence of chronic lung disease and also

4 1308 Graham et al Table IV Intrapartum assessment Controls Cases P value OR, 95% CI Absent end diastolic flow 7/150 (5%) 7/150 (5%) , Nonreassuring FHR tracing 35/150 (23%) 34/150 (23%) , Umbilical arterial ph 7.27 G G Umb art BE (mmol/l) ÿ2.1 G 2.7 ÿ3.0 G ph! 7.0 2/122 (2%) 3/107 (3%) , BE! ÿ12 mmol/l 4/121 (3%) 6/106 (6%) , FHR, Fetal heart rate, BE, base excess, Umb art, umbilical arterial; OR, odds ratio; CI, confidence interval. Table V Infectious risk Controls Cases P value OR, 95% CI Clinical chorio. 29 (19%) 28 (19%) , Histologic chorio. 80 (57%) 74 (54%) , Histologic funisitis 43 (31%) 39 (29%) , Positive urine culture 11 (7%) 19 (13%) , Positive blood culture 28 (19%) 43 (29%).036* 2.0, Age at culture (d) 17.8 G G Positive culture,!7 d 7/28 (25%) 6/43 (14%).24 Positive CSF culture 9 (6%) 26 (17%).002* 3.83, Age at culture (d) 16.1 G G Positive culture,!7 d 3/9 (33%) 1/26 (3.8%).02* Positive trachea culture 14 (9%) 33 (22%).003* 3.11, Age at culture (d) 30.1 G G Positive culture,!7 d 0/14 2/33 (6.1%).35 Chorio, Chorioamnionitis; CSF, cerebrospinal fluid. Case positive blood cultures: Staphylococcus coagulase negative, 17; Enterobacter, 4; Candida, 4; Streptococcus viridans, 2; Citrobacter, 2; Klebsiella pneumoniae, 2; Staph. aureus, 1;Escherichia coli 1; group B Streptococcus, 1; other 9. Case positive CSF cultures: Staphylococcus coagulase negative, 19; Enterococcus,2;Serratia,1;Enterobacter,1;Strep. group D, 1; Candida, 1; other, 1. Case positive tracheal cultures: Staph. aureus, 8; Enterobacter, 6; K. pneumoniae, 4; Pseudomonas aeruginosa, 4; E. coli, 3; Staph. coagulase negative, 3; Serratia, 2; other, 4. intraventricular hemorrhage (IVH), a known risk factor for white matter injury and cerebral palsy. Neither umbilical arterial Doppler studies showing absent end diastolic flow prior to delivery nor nonreassuring fetal heart rate tracing during labor were predictive of which fetuses would develop cerebral white matter injury (Table IV). The mean umbilical arterial ph and base excess were very similar for cases and controls. The incidence of an umbilical arterial ph less than 7.0 and a base excess less than e12.0 mmol/l, cutoffs that have been linked with an increased risk of neurologic morbidity, were similar between cases and controls and occurred in only 3%-6% of neonates with cerebral white matter injury. Neither clinical nor histologically confirmed chorioamnionitis was associated with an increased risk of cerebral white matter injury; however, culture-positive neonatal infections were associated with an increased risk (Table V). A positive neonatal blood culture was associated with a 2.0-fold increased risk for cerebral white matter injury, a positive tracheal culture with a 3.1-fold increase, and a positive cerebrospinal fluid (CSF) culture with a 3.8-fold increase. Of the patients with positive blood cultures, 71% of controls and 58% of cases had histologic chorioamnionitis (P =.26). Of the patients with positive CSF cultures, 56% of controls and 73% of cases had histologic chorioamnionitis (P =.42). Of the patients with positive tracheal cultures, 79% of controls and 52% of cases had histologic chorioamnionitis (P =.11). A conditional (fixed effects) logistic regression model was created to determine which of these variables were predictive of the development of neonatal cerebral white matter injury (Table VI). The pseudo-r 2 for this model was 0.31, indicating the proportion of the variance in the dependent variable (cerebral white matter injury) explained by all the specified independent variables. Multiple gestation, IVH, and positive tracheal cultures were significantly associated with the development of cerebral white matter injury. Positive CSF cultures nearly reached the level of statistical significance (P =.052). Comment Our study found a significantly higher incidence of multiple gestations among neonates with cerebral white matter injury. An association between multiple gestation and

5 Graham et al 1309 cerebral palsy has been established for some time, which is not surprising because preterm delivery is strongly associated with cerebral palsy and multiple gestations are at much higher risk for preterm birth. In very low-birthweight infants, some investigators 6 have found no difference in the rates of cerebral palsy between singletons and twins. Others 7 have found multiple pregnancy to be a risk factor for cerebral palsy at all gestational ages, and our study agrees with this, showing a higher incidence of multiple gestations with white matter injury even when matched by gestational age. Monozygosity, and particularly monochorionicity, have been strongly linked with an increased risk of neurologic impairment; however, the incidence of monochorionic placentation was roughly the same in both the case and control groups in our study. Metabolic acidemia must be present at birth to establish that a potentially damaging intrapartum hypoxic event is a cause of neurologic injury. 8 Even though normal preterm infants have a mean umbilical arterial ph of 7.27 G 0.7, 9 there is not an increase in neonatal neurologic morbidity until the umbilical arterial ph is less than Even when this ph threshold of less than 7.0 is used to define significant acidemia, most newborns in this category will be neurologically normal with no apparent morbidity. 11 Before the ph drops, buffer base must be consumed, so the base excess may be a more sensitive indicator of the fetus developing significant metabolic acidosis. There is not an increase in neonatal neurologic morbidity until the base excess falls to less than e12.0 mmol/l. 12 Because the cerebral white matter in preterm infants receives such low blood flow, compared with the cortex, and has a minimal margin of safety, if there were intrapartum hypoxia-ischemia severe enough to lead to white matter injury, this would be detected in the umbilical arterial gas. This study shows that significant metabolic acidosis occurs in only 6% of preterm neonates diagnosed with cerebral white matter injury and is not significantly different from the incidence of 3% in gestational agematched controls without brain injury. This demonstrates that intrapartum hypoxia-ischemia as manifested by metabolic acidosis is a rare cause of brain injury in preterm neonates. This is in agreement with other data such as a case-control study of all 183 cases of cerebral palsy in western Australia from 1975 to 1980, which found that intrapartum injury was a likely cause of cerebral palsy in only 4.9% of cases and a possible cause in only an additional 3.3% of cases. 13 Our univariate analysis showed a significantly higher incidence of positive blood, CSF, and tracheal cultures in preterm infants with brain injury. The brain-injured group also had a higher incidence of chronic lung disease. For the 52-73% of neonates with cerebral white matter injury and histologic chorioamnionitis present at the time of delivery, this infection could have arisen any time during the pregnancy, but for the other 48-27% with no Table VI Conditional logistic regression of variables associated with the presence of white matter injury Coefficient SE P value 95% CI Multiple gestation * 0.15, 1.97 Chronic lung ÿ0.33, 1.25 disease Death ÿ ÿ2.30, 0.41 IVH !.001* 0.87, 2.23 Positive blood ÿ ÿ0.88, 0.81 culture Positive CSF culture ÿ0.01, 2.11 Positive trachea culture * ÿ2.30, 0.41 IVH, Intraventricular hemorrhage; CSF, cerebrospinal fluid. evidence of histologic chorioamnionitis at delivery, the infection may have arisen after birth. Conditional logistic regression showed positive tracheal cultures, and very nearly positive CSF cultures, to be associated with injury. Infection is a risk factor for the development of cerebral palsy, and this may be mediated by proinflammatory cytokines such as interleukin-1b, interleukin-6, and tumor necrosis factor-a. Although increased levels of cytokines have been found in term infants who later developed cerebral palsy, 14 the evidence of a link between infection and cerebral palsy in preterm infants is inconsistent. A study of 64 neonates later diagnosed with cerebral palsy and 107 controls born at less than 32 weeks did not find that neonatal blood levels of inflammatory cytokines could distinguish those infants who were later diagnosed with cerebral palsy. 15 However, a recent meta-analysis found that chorioamnionitis was associated with both cerebral palsy and cystic PVL in preterm infants. 16 Increased amniotic fluid cytokine levels have been associated with an increased risk of neonatal brain and lung injury. 17,18 An association has been shown between umbilical cord blood cytokine concentrations and the development of cerebral white matter lesions. 19 High levels of tumor necrosis factor-a have been found in neonatal brains with PVL. 20 A cytokine hypothesis has been put forward, speculating that a number of different insults, including infection, hypoxia-ischemia, reperfusion, and toxin-mediated injury, may produce cytokines that act as a final common pathway for injury to the central nervous system. 21 Antenatal steroid administration has been associated with a 56% reduction in the risk of PVL with IVH and a 58% reduction in the risk of PVL alone. 22 Antenatal steroids may decrease the systemic inflammatory response associated with infection and decrease the harmful effects of cytokines on the developing oligodendrocyte. The incidence of PVL is higher in infants who sustain IVH. The hemorrhage provides a rich source

6 1310 Graham et al of iron for the generation of reactive oxygen species. Oligodendrocyte progenitor cells have a delay in the development of antioxidant defenses, especially glutathione peroxidase and catalase, and are deficient in the capability of handling free radicals. 3 These enzymes are involved in the detoxification of hydrogen peroxide, and when they fail or are overwhelmed, hydrogen peroxide accumulates, and in the presence of Fe 2C, the Fenton reaction produces the deadly hydroxyl radical. 3 Inflammatory cytokines may link both IVH and white matter injury. In conclusion, this study found that intrapartum hypoxia-ischemia, as manifested by metabolic acidosis, is rarely associated with preterm neonatal cerebral white matter injury. The incidence of severe metabolic acidosis was 3%-6% in the brain-injured preterm neonates, which was not significantly different from the incidence of 3% in gestational age-matched controls. Although histologic chorioamnionitis and funisitis were not associated with cerebral white matter injury, culture-positive neonatal infection was. The inflammatory cytokine response may possibly link neonatal infection with the development of cerebral white matter injury. References 1. Leviton A, Paneth N. White matter damage in preter newbornsdan epidemiologic perspective. Early Hum Dev 1990;24: Borch K, Greisen G. Blood flow distribution in the normal human preterm brain. Pediatr Res 1998;43: Volpe JJ. Neurobiology of periventricular leukomalacia in the premature infant. Pediatr Res 2001;50: Staudt M, Pavlova M, Bohm S, Grodd W, Krageloh-Mann I. Pyramidal tract damage correlates with motor dysfunction in bilateral periventricular leukomalacia (PVL). Neuropediatrics 2003; 34: Nelson KB, Grether JK. Can magnesium sulfate reduce the risk of cerebral palsy in very low birthweight infants? Pediatrics 1995; 95: Dunin-Wasowicz D, Rowecka-Trzebicka K, Milewska-Bobula B, Kassur-Siemienska B, Bauer A, Idzik M, et al. Risk factors for cerebral palsy in very low-birthweight infants in the 1980s and 1990s. J Child Neurol 2000;15: Williams K, Hennessy E, Alberman E. Cerebral palsy: effects of twinning, birth weight, and gestational age. Arch Dis Child Fetal Neonatal Ed 1996;75:F American College of Obstetricians and Gynecologists, American Academy of Pediatricians. Neonatal encephalopathy and cerebral palsy: defining the pathogenesis and pathophysiology. Washington, DC: Riley RJ, Johnson JWC. Collecting and analyzing cord blood gases. Clin Obstet Gynecol 1993;36: Goldaber KG, Gilstrap LC III, Leveno KJ, Dax JS, McIntire DD. Pathologic fetal acidemia. Obstet Gynecol 1991;78: Goodwin TM, Belai I, Hernandez P, Durand M, Paul RH. Asphyxial complications in the term newborn with severe umbilical acidemia. Am J Obstet Gynecol 1992;167: Low JA, Lindsay BG, Derrick EJ. Threshold of metabolic acidosis associated with newborn complications. Am J Obstet Gynecol 1997;177: Stanley FJ, Blair E. Why have we failed to reduce the frequency of cerebral palsy? Med J Aust 1991;154: Nelson KB, Dambrosia JM, Grether JK, Phillips TM. Neonatal cytokines and coagulation factors in children with cerebral palsy. Ann Neurol 1998;44: Nelson KB, Grether JK, Dambrosia JM, Walsh E, Kohler S, Satyanarayana G, et al. Neonatal cytokines and cerebral palsy in very preterm infants. Pediatr Res 2003;53: Wu YW. Systematic review of chorioamnionitis and cerebral palsy. Ment Retard Dev Disabil Res Rev 2002;8: Dammann O, Leviton A. Role of the fetus in perinatal infection and neonatal brain damage. Curr Opin Pediatr 2000;12: Yoon BH, Romero R, Jun JK, Park KH, Park JD, Ghezzi F, et al. Amniotic fluid cytokines (interleukin-6, tumor necrosis factoralpha, interleukin-1 beta, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia. Am J Obstet Gynecol 1997;177: Yoon BH, Jun JK, Romero R, Park KH, Gomez R, Choi JH, et al. Amniotic fluid inflammatory cytokines (interleukin-6, interleukin- 1-beta, and tumor necrosis factor-alpha), neonatal brain white matter lesions, and cerebral palsy. Am J Obstet Gynecol 1997; 177: Kadhim H, Tabarki B, Verellen G, De Prez C, Rona AM, Sebire G. Inflammatory cytokines in the pathogenesis of periventricular leukomalacia. Neurology 2001;56: Dammann O, Leviton A. Infection remote from the brain, neonatal white matter damage, and cerebral palsy in the preterm infant. Semin Pediatr Neurol 1998;5: Canterino JC, Verma U, Visintainer PF, Elimian A, Klein SA, Tejani N. Antenatal steroids and neonatal periventricular leukomalacia. Obstet Gynecol 2001;97:135-9.