Cerebral White Matter Damage Research Articles

Changes in white matter diffusion anisotropy in adolescents born prematurely

Being born with very low birth weight (VLBW, birth weight ≤1500 g) or small for gestational age (SGA) carries an increased risk of cerebral white matter damage. The reduced cognitive and motor skills these two groups exhibit suggest that the early injuries to white matter persist into adolescence. White matter integrity was assessed using voxel-wise statistical analysis of fractional anisotropy (FA) maps between three groups of adolescents at age 15; the VLBW group ( n = 34), the SGA group ( n = 42) and a control group with normal birth weight ( n = 47). The FA maps were normalized to a study specific template and group differences were assessed using an analysis of covariance with gender as a confounder (FDR-corrected P < 0.05). The main finding is that the VLBW group has significantly reduced FA values in several white matter regions, including the corpus callosum, internal capsule and superior fasciculus compared to the control group. Some of the observed reduction in anisotropy, particularly that observed in the corpus callosum, may have been caused by inaccurate spatial normalization, but this can only explain 30% of the area with reduced anisotropy. Analysis of the eigenvalues of the diffusion tensor show that the reduced FA values in the VLBW group is primarily due to an increase in the two lowest eigenvalues of the diffusion tensor. We speculate that this may be caused by reduced myelination. For the SGA group, we find no statistically significant differences in anisotropy compared to the control group.

Acute cerebral white matter damage in lethal salicylate intoxication

A 34-year-old oligophrenic woman was admitted in comatose state with marked tachypnea. History revealed the oral ingestion of a large amount of acetylsalicylate to attenuate ear pain within the preceding 3 days. Laboratory investigations showed a toxic concentration of serum salicylate (668 mg/l, toxic range above 200 mg/l) and metabolic acidosis. Oxygenation, blood pressure, electrocardiography, echocardiography and CT of thorax and brain were normal. The patient was intubated, fluid and bicarbonate was given intravenously. Six hours after admission asystolia refractory to resuscitation led to death. Autopsy showed venous congestion of the brain, cardiac dilatation and pulmonary edema. Brain histopathology showed myelin disintegration and caspase-3 activation in glial cells, whereas, grey matter changes were sparse. Acute white matter damage is suggested to be the substrate of cerebral dysfunction in salicylate intoxication and possible mechanisms are discussed.

Spatial heterogeneity in oligodendrocyte lineage maturation and not cerebral blood flow predicts fetal ovine periventricular white matter injury.

Although periventricular white matter injury (PWMI) is the leading cause of chronic neurological disability and cerebral palsy in survivors of premature birth, the cellular-molecular mechanisms by which ischemia-reperfusion contributes to the pathogenesis of PWMI are not well defined. To define pathophysiologic relationships among ischemia, acute cerebral white matter damage, and vulnerable target populations, we used a global cerebral ischemia-reperfusion model in the instrumented 0.65 gestation fetal sheep. We developed a novel method to make repeated measurements of cerebral blood flow using fluorescently labeled microspheres to resolve the spatial heterogeneity of flow in situ in three-dimensional space. Basal flow in the periventricular white matter (PVWM) was significantly lower than in the cerebral cortex. During global cerebral ischemia induced by carotid occlusion, flow to all regions was reduced by nearly 90%. Ischemia of 30 or 37 min duration generated selective graded injury to frontal and parietal PVWM, two regions of predilection for human PWMI. Injury was proportional to the duration of ischemia and increased markedly with 45 min of ischemia to extensively damage cortical and subcortical gray matter. Surprisingly, the distribution of PVWM damage was not uniform and not explained by heterogeneity in the degree of white matter ischemia. Rather, the extent of white matter damage coincided with the presence of a susceptible population of late oligodendrocyte progenitors. These data support that although ischemia is necessary to generate PWMI, the presence of susceptible populations of oligodendrocyte progenitors underlies regional predilection to injury.

Maternal Exposure to LPS Induces Hypomyelination in the Internal Capsule and Programmed Cell Death in the Deep Gray Matter in Newborn Rats

Epidemiologic and experimental findings implicate maternal infection in the etiology of injury to brain white matter, which may lead to cerebral palsy in preterm newborns. In the present study, inflammation and brain damage in 1- and 7-d-old rats were investigated after maternal inflammation. Intraperitoneal injection of 300 microg/kg of Escherichia coli lipopolysaccharide was administered to pregnant Wistar rats at d 19 and 20 of gestation (LPS group). Control females received a saline injection. Proinflammatory cytokines IL-1beta, tumor necrosis factor-alpha, and IL-6 expression in the fetal brain were determined by reverse transcription quantitative polymerase chain reaction. Brain injury was examined in 16-mum coronal brain sections by GFAP, MBP, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Expression of IL-1beta was significantly increased 3 d after maternal administration (P1). A significant increase in cell death occurred at P1 and P7 in specific brain areas, i.e. in the subventricular striatal zone at P1, and in 1) the periventricular striatum, 2) the periventricular white matter, and 3) the germinative ventricular zone at P7. We also observed typical astrogliosis and strong hypomyelination in the external and internal capsule in the LPS group at P7. These results demonstrate that maternal LPS treatment induces persistent fetal inflammatory reactions associated with significant white matter injury in progeny at P1 and P7. This model should be relevant for the study of the pathophysiological mechanisms involved in cerebral white matter damage in preterm human newborns and in the development of therapeutic strategies.

Brain Injury in Congenital Heart Disease

Mortality rates for virtually all forms of congenital heart disease have declined with dramatic advances in medical, transcatheter, and surgical therapies. 1 As the population of congenital heart disease survivors has burgeoned, however, their long-term functional morbidities have become the focus of increasing concern. Among the foremost morbidities are adverse neurodevelopmental outcomes, with their profound personal and societal costs. 2 Article p 280 Neurological and developmental outcomes of congenital heart patients are influenced by many factors, both innate and acquired, with cumulative effects. Genetic syndromes such as trisomy 21 or 22q11 microdeletion3–5 may affect both the heart and the brain. Cerebral dysgenesis is reported to occur in 10% to 29% of children with congenital heart disease in autopsy series, with the incidence varying by lesion 6–8 ; findings may range from microdysgenesis to gross abnormalities such as agenesis of the corpus callosum, incomplete operculization, and microcephaly. During fetal life, congenital heart lesions may be associated with changes in cerebrovascular blood flow distribution and resistance. For example, fetuses with hypoplastic left heart syndrome, whose cerebral perfusion is supplied retrograde through the ductus arteriosus, have lower cerebrovascular resistance than normal. 9 Postnatal neurodevelopmental risk factors may derive from the sequelae of congenital heart disease itself—eg, chronic severe hypoxemia, failure to thrive, arrhythmias with cardiac arrest or hypotension—or from the procedures used for cardiac correction or palliation.10 Neuropathological studies have revealed both focal and diffuse infarction. Focal infarction has been ascribed to thromboembolic events, whereas a diffuse pattern of cerebral injury has been attributed to hypotension and hypoperfusion.11 More recent neuropathological data in infants who underwent reparative or palliative cardiac surgery reveals that not only are these children at increased risk of gray matter injury but also nascent white matter is at risk. For instance, in a series of infants who died after cardiac surgery, cerebral white matter damage (periventricular leukomalacia or diffuse white matter gliosis) was the most significant lesion in terms of severity and incidence. 12 Similarly, MRI performed in patients with congenital heart disease after surgery has revealed findings consistent with both gray matter injury and widely distributed white matter injury. 13 Risk factors for brain injury during infant heart surgery have been particularly well studied. Cardiopulmonary bypass carries the risks of particulate and gaseous microemboli, macroemboli, and hypoperfusion with accompanying diffuse ischemia/reperfusion injury. In neonates and infants undergoing surgery with use of hypothermic bypass techniques, the risk of brain injury may be influenced by perfusion variables such as the duration of total circulation arrest,14 the depth of hypothermia, 15 the rate and duration of core cooling, 16 pH management during core cooling (alpha-stat versus pH stat), 17,18 and the level of hemodilution. 19 Disruption of cerebral vasoregulation in the early postoperative period renders the brain more vulnerable to hemodynamic instability. 20–22 After infant heart surgery, longer length of stay in the intensive care unit or hospital is associated with worse neurodevelopmental outcome on mid-term follow-up. 23,24

The adaptive immune response in neonatal cerebral white matter damage

Hypotheses that inflammation contributes to neonatal cerebral white matter damage have evolved over the last three decades. The latest, expanded here, suggests that the adaptive immune system contributes to the intensity and duration of the processes that result in damage to cerebral white matter in the fetus and newborn. We propose several mechanisms by which fetal T lymphocytes could be activated during fetal exposure to infection. These include specific recognition of bacterial antigens, specific recognition of autoantigens, polyclonal activation by Toll-like receptors, and bystander activation by cytokines.

Hypoxic-ischemic brain injury in infants with congenital heart disease dying after cardiac surgery

Cardiac surgery for congenital heart disease is performed increasingly earlier in infancy, including in the neonatal period. With increased survival of infants, there is growing concern about the long-term neurological sequelae of hypoxic-ischemic injury due to congenital heart disease itself prior to surgery, corrective surgery with the use of low-flow cardiopulmonary bypass (CPB) and/or deep hypothermic circulatory arrest (DHCA), and/or unstable hemodynamic factors postoperatively. In analyzing the neuropathology of 38 infants dying after cardiac surgery, we tested a set of questions related to the severity and patterns of brain injury, CPB, DHCA, and age of the infants at the time of surgery. In all infants dying after cardiac surgery, irrespective of the modality, cerebral white matter damage [periventricular leukomalacia (PVL) or diffuse white matter gliosis] was the most significant lesion in terms of severity and incidence, followed by a spectrum of gray matter lesions. There was no significant association between the duration of deep hypothermic circulatory arrest and the degree of severity of overall brain injury, and the pattern of brain injury was similar irrespective of the modality of cardiac surgery. There was no significant association between the age at the time of surgery (neonatal versus postneonatal) and the severity of overall brain injury. The patterns of brain injury were not age-related in the limited time-frame analyzed, except that infants who developed acute PVL after both closed and DHCA/CPB surgery (14/38 infants, 34%) were significantly younger at death (median age 13.0 days) compared to unaffected infants (median age at death 42.5 days) (P=0.031). This observation suggests that neonatal (<30 postnatal days), but not postneonatal (>30 postnatal days), brains are at risk for acute PVL, and likely reflects the vulnerability of immature (pre-myelinating) white matter to hypoxia-ischemia.

Variable interpretation of ultrasonograms may contribute to variation in the reported incidence of white matter damage between newborn intensive care units in New Zealand

Background: The incidence of cerebral white matter damage reported to the Australian and New Zealand Neonatal Network (ANZNN) varies between neonatal intensive care units (NICUs). Hypothesis: Differences in the capture,...

319 Maternal LPS Treatment Induces Apoptosis and White Matter Lesions in Newborn Rat Brain

Introduction: Periventricular leucomalacia is a white matter lesion characterized by hypomyelination that occurs in preterm human newborn. Maternal inflammation seems to be a major cause of this lesion.Objective: Our aim was to study brain damage after maternal inflammation in newborn rats on P1 and P7.Methods: Inflammation was caused by Escherichia coli lipopolysaccharide administration to pregnant rats at days 19 and 20 of gestation (LPS group, N=8). Control rats got a saline injection (N=8). Neonats were studied at P1 (7 from LPS group, 8 from control) and P7 (8 from LPS group, 7 from control). Myelination was estimated using MBP immunohistochemistry and apoptosis using Terminal transferase assay (TUNNEL) and Caspase-3 immunohistochemistry. Brain injury was examined on 16μm thickness coronal brain sections.Results: At P1, a 114% increase was observed in the number of caspase-3 positive cells in the subventricular striatal zone (40 ± 5 cells per field in the LPS group vs 21 ± 6 in controls, p<0.05). At P7, significant increase of apoptotic cells was found in specific brain areas, i.e. (1) the periventricular striatum (TUNEL: 24 ± 3 cells per field in the LPS group vs 14 ± 2 in controls, p<0.05; Caspase-3: 18 ± 2 vs 8 ± 1, p=0.001), (2) the periventricular white matter (Caspase-3: 12 ± 3 vs 4 ± 1, p<0.05), (3) the germinative ventricular zone: (Caspase-3: 22 ± 4 vs 9 ± 2, p<0.05). At P7, we also observed a strong hypomyelination in 6 out of 8 animals from the LPS group in the external and internal capsules.Conclusions: These results indicate that maternal LPS treatment induces apoptosis associated with strong white matter injury at P7. This model could be relevant for the study of the pathophysiological mechanisms involved in cerebral white matter damage in preterm human newborn.

195 Prenatal Inflammation, Brain Damage and Two-Year Neurologic Outcome in Very Preterm Infants

Background: Intrauterine inflammation has been associated with preterm birth and with fetal inflammatory response affecting various organs including immature brain. Prenatal etiology has been defined in a majority of term infants with later neurologic sequela but only in a minority of very preterm infants. Risk factors of cerebral white matter damage (WMD) may differ between term and preterm infants.Objective: To examine whether prenatal inflammation serves as a risk factor of WMD and poor neurologic and neurocognitive outcome in infants born before 32 weeks of gestation with birth weight under 1000 g.Design/Methods: A regional cohort of 61 infants born between 1998 and 2002 were enrolled in a prospective follow-up study. Medical records during hospitalization were observed. Placental pathology was examined. Sera from umbilical cord blood were analyzed for proinflammatory cytokines (IL-1 alpha, IL-1 beeta, IL-6, IL-8, TNF-alpha). Serial brain ultrasound examinations and magnetic resonance imaging were performed. Neurologic and neurocognitive outcome were assessed at two years of corrected age. The study was approved by the ethics committee of Oulu University Hospital.Results: Histologic chorioamnionitis (HCA) and high IL-6 predicted spontaneous preterm labor with high accuracy. High IL-6 and IL-8 associated with HCA. Intraventricular hemorrhage grade 2 to 3 was predicted by HCA (OR 8.1, 95%CI 1.6 - 42.5). Neither HCA or the cytokines directly associated with WMD, poor neurologic, or poor neurocognitive outcome. However, HCA and concomitant placental perfusion defect increased the risk of poor neurologic outcome (OR 15.2, 95%CI 1.3 - 181) and together with WMD additively predicted poor outcome. Several clinical factors describing cardiopulmonary instability after birth associated with WMD and with poor neurologic outcome.Conclusions: HCA and fetal cytokine response are not risk factors of WMD in very preterm extremely low birth weight infants. Present results support the theory of a complexity of pathogenetic mechanisms leading to poor neurologic outcome.

White Matter Injury Following Prolonged Free Radical Formation in the 0.65 Gestation Fetal Sheep Brain

Free radicals seem to be involved in the development of cerebral white matter damage after asphyxia in the premature infant. The immature brain may be at increased risk of free radical mediated injury, as particularly the preterm infant has a relative deficiency in brain antioxidants systems, such as superoxide dismutase and glutathione peroxidase. In vitro studies show that immature oligodendrocytes express an intrinsic vulnerability to reactive oxygen species and free radical scavengers are able to protect immature oligodendrocytes from injury. The aim of this study was to examine the formation of ascorbyl radicals as a marker of oxidative stress in the preterm brain in association with cerebral white matter injury after intrauterine asphyxia. Fetal sheep at 0.65 gestation were chronically instrumented with vascular catheters and an occluder cuff around the umbilical cord. A microdialysis probe was placed in the periventricular white matter. Fetal asphyxia was induced by occlusion of the umbilical cord for 25 min (n = 10). Microdialysis samples were collected for 72 h and analyzed for ascorbyl radicals using electron spin resonance. Five instrumented fetuses served as controls. Three days after the insult, fetal brains were examined for morphologic injury. Umbilical cord occlusion resulted in prolonged and marked increase in ascorbyl radical production in the brain in connection with white matter injury, with activation of microglia cells in periventricular white matter and axonal injury. These data suggest that reperfusion injury following asphyxia in the immature brain is associated with marked free radical production.

Can obstetric care provide further improvement in the outcome of preterm infants?

Reducing the incidence of cerebral damage in preterm infants has become a major objective of perinatal medicine. Recent studies have shown that intrapartum hypoxia is implicated in only 10% of cases, whereas prenatal factors are significantly linked with such damage. The main risk factors associated with cerebral palsy are preterm birth, multiple pregnancy, intrauterine infection, serious hypoxaemic and haemodynamic disorders and, possibly, thrombophilic disorders. Recent progress in recognition of the pathogenesis of cerebral white matter damage has underlined the roles of cytokines, including interleukins 1 and 6 and tumour necrosis factor alpha (TNFα), and of a massive release of glutamate, which leads to the excitotoxic cascade. Three measures have had demonstrable benefits in improving neonatal outcome in preterm infants: a policy of prenatal transfers to tertiary level care units, antenatal corticosteroid therapy, and administration of antibiotics to women with preterm premature rupture of membranes. The influence of tocolysis is usually considered to be relatively minor, but further studies in very preterm pregnancies may be required. The recent development of tocolytics without major adverse effects will probably facilitate such studies. The mode of delivery of early preterm infants is another important area of controversy. Recent studies have suggested that a policy of elective caesarean section in PPROM and in the case of breech presentation of fetuses weighing less than 1000–1500 g would be beneficial. Lastly, in vitro and animal studies have shown that several pharmacological agents can prevent white matter disease by interacting with cytokine and the excitotoxic cascade. This will probably constitute an important area of research in the future.

Brain development during fetal life: influences of the intra-uterine environment*1

The intrauterine environment can significantly affect fetal brain development. Here we review our recent findings using animal models that mimic adverse intrauterine conditions which could exist during human pregnancy. We have focused on effects of both acute and chronic hypoxic and inflammatory insults. Relatively brief periods of hypoxemic compromise can have significant effects on the fetal brain causing neuronal loss and cerebral white matter damage. Subtle brain injury can occur, for example to a particular class of neuron, and this can have a significant effect on the function of a specific system. Chronic mild placental insufficiency can result in long term deficits in neuronal connectivity affecting function postnatally as demonstrated in the auditory and visual systems. Repeated acute exposure to an inflammatory agent results in diffuse subcortical white matter damage and in some cases periventricular necrosis. We have demonstrated that the timing and severity of these prenatal insults are determinants of the outcomes, in terms of the severity of the damage and the regions of the brain affected.

Association between apolipoprotein E e4 allele and arteriosclerosis, cerebral amyloid angiopathy, and cerebral white matter damage in Alzheimer’s disease

Objective: To investigate the association between white matter damage, as evidenced by myelin loss (ML), the extent of cerebral amyloid angiopathy (CAA), or arteriosclerosis (Art), and apolipoprotein E (ApoE) e4...

Brain development during fetal life: influences of the intra-uterine environment

The intrauterine environment can significantly affect fetal brain development. Here we review our recent findings using animal models that mimic adverse intrauterine conditions which could exist during human pregnancy. We have focused on effects of both acute and chronic hypoxic and inflammatory insults. Relatively brief periods of hypoxemic compromise can have significant effects on the fetal brain causing neuronal loss and cerebral white matter damage. Subtle brain injury can occur, for example to a particular class of neuron, and this can have a significant effect on the function of a specific system. Chronic mild placental insufficiency can result in long term deficits in neuronal connectivity affecting function postnatally as demonstrated in the auditory and visual systems. Repeated acute exposure to an inflammatory agent results in diffuse subcortical white matter damage and in some cases periventricular necrosis. We have demonstrated that the timing and severity of these prenatal insults are determinants of the outcomes, in terms of the severity of the damage and the regions of the brain affected.

Coagulation, inflammation, and the risk of neonatal white matter damage.

Indicators of coagulation activation are sometimes increased in the blood of newborns and adults who have a systemic inflammatory response. These coagulation factors have the ability to exacerbate inflammation, which in turn can promote coagulation. Therapies directed solely at coagulation factors and therapies directed solely at inflammation factors have not proved effective in reducing mortality in adults with a systemic inflammatory response syndrome and multi-organ dysfunction (SIRS/MOD). On the other hand, the only therapy that has reduced mortality in SIRS/MOD is activated protein C, which has both anti-coagulation and anti-inflammatory effects. This and other observations support the view that activated coagulation factors enhance inflammation. Since newborns at risk of cerebral white matter damage and cerebral palsy are more likely than their peers to have a systemic inflammatory response, which is sometimes accompanied by elevated blood levels of coagulation factors, we suggest that activated coagulation factors contribute to the occurrence of cerebral white matter damage by exacerbating inflammatory phenomena, rather than by occluding cerebral blood vessels.

Bronchopulmonary dysplasia is not associated with ultrasound-defined cerebral white matter damage in preterm newborns.

Bronchopulmonary dysplasia (BPD) and cerebral white matter damage (WMD) are neonatal disorders that occur most commonly in those who are born much before term. In a large multicenter database, we sought to determine whether the two disorders occur together more frequently than expected and whether BPD and other neonatal respiratory characteristics are more common among infants who develop ultrasound-defined WMD than among those who do not. In a sample of 904 infants who were born before the 30th week of gestation and survived until 36 wk postmenstrual age, we did not find a co-occurrence of BPD and WMD above what would be expected by chance. Confounding does not seem to account for this lack of association between WMD and BPD. In conclusion, our findings do not support the hypothesis that BPD contributes to the occurrence of sonographically defined WMD.

Glyceraldehyde 3-phosphate dehydrogenase and endothelin-1 immunoreactivity is associated with cerebral white matter damage in dentatorubral-pallidoluysian atrophy.

DRPLA is a rare neurodegenerative disorder caused by CAG triplet elongation on chromosome 12p. In addition to neurodegeneration of both the dentatorubral and pallidoluysian systems, there is cerebral white matter damage, especially in older cases. Intracellular accumulation of DRPLA protein is widespread in the central nervous system, and DRPLA protein has been shown to immobilize glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which regulates glycolysis and controls mRNA of tissue-type plasminogen activator (tPA) in tissue restoration. However, little is known about the pathogenesis regarding the formation of cerebral white matter damage in DRPLA. Therefore, the pathology of this damage was investigated by examining markers of glycolysis and related processes. Nine clinically and pathologically confirmed DRPLA cases were used in the present study. CAG triplet elongation on chromosome 12p was confirmed in all cases where tissue was available for genotyping (seven cases). PAS and immunohistochemistry with antibodies to GFAP, GAPDH and endothelin-1 were used to demonstrate astrocytosis. The polysaccharides storage state with PAS-positive astrocytes was detected in seven cases. GAPDH- and endothelin-1-positive endothelium and astrocytes were observed in two cases with GFAP-positivity. Based on the biochemical process together with the present results, GAPDH and endothelin-1 immunoreactivity is associated with this damage and the mismetabolism of polysaccharides caused by CAG triplet elongation on chromosome 12p may contribute to the formation of the cerebral white matter damage in DRPLA.

Adult stroke and perinatal brain damage: like grandparent, like grandchild?

Biomarkers of inflammation are found in the circulation of adults who have had a stroke. Although these biomarkers may, in part, be indicators of damage, some appear to contribute to damage. Similar biomarkers are found in newborns with cerebral white matter damage or at risk of cerebral palsy. Can we learn about the pathogenesis of neonatal white matter damage from what has been learned about the inflammatory correlates of adult stroke? We discuss relevant findings about systemic inflammatory markers in adult stroke and relate this information to our current understanding of cerebral white matter damage in newborns, especially those born at an extremely low gestational age. We also describe desirable characteristics of future studies of perinatal brain damage that involve measurements of systemic biomarkers.

Antenatal mycoplasma infection, the fetal inflammatory response and cerebral white matter damage in very-low-birthweight infants.

We address the question as to whether Ureaplasma urealyticum or Mycoplasma hominis, cultured from the placenta of very-low-birthweight (VLBW) infants, are associated with an increased risk of (a) fetal vasculitis and (b) ultrasonographic cerebral white matter echolucency. The sample consisted of 464 VLBW infants for whom (i) the surface of the chorion was cultured for U. urealyticum and M. hominis; (ii) the placenta was examined histologically; and (iii) a cranial ultrasound scan was obtained close to days 1, 7 or 21. Infants with echolucency were compared with controls in univariable and stratified analyses and in multivariable logistic regressions. Fifty-three per cent of placentas from infants with fetal vasculitis harboured U. urealyticum compared with 18% of controls (P </= 0.001). M. hominis was present in 14% of cases of fetal vasculitis and in 2% of controls (P </= 0.001). Among cases of echolucency, 22% had U. urealyticum compared with 30% of controls (P = 0.33), whereas 17% of these cases and 5% of controls had M. hominis (P = 0.08). Our findings support the hypothesis that U. urealyticum in the placenta of VLBW infants contributes to the fetal inflammatory response without contributing to white matter damage. Our finding that M. hominis in the placenta was associated with a not quite significant threefold risk increase for echolucency deserves further investigation.