globally , preterm birth ( defined as birth at < 37  weeks of gestation ) affects around 11% of deliveries ( blencowe et al . , 2013 ) and it is closely associated with cerebral palsy , cognitive impairment and neuropsychiatric disease in later life ( kerr - wilson et al . , 2012 ; moore et al . , 2012 ) .
individuals born preterm are more likely to experience behavioral and emotional disturbance ( clark et al . , 2008 ; elgen et al . ,
2012 ) , autistic spectrum disorder ( johnson et al . , 2010 ) , attention deficit hyperactivity disorder ( bhutta et al . , 2002 ) and psychiatric disease , and these problems persist into adulthood ( crump et al . , 2010 ; halmoy et al . , 2012 ; moster et al . , 2008 ; nosarti et al . ,
white matter disease is a core feature of the encephalopathy of prematurity and it is associated with reduced connectivity of neural circuitry ( ball et al . , 2013 ;
boardman et al . , 2006 ; fischi - gomez et al . , 2014 ; pandit et al . , 2014 ; woodward et al
antenatal magnesium sulfate ( mgso4 ) is often given to women expected to deliver preterm because it is associated with reduced rates of cerebral palsy ( doyle et al .
. however , there are uncertainties about its long - term effects , and studies have not been designed or powered to investigate its influence on non - motor functions ( chollat et al . , 2014 ; doyle et al . , 2014 )
biomarkers of tissue integrity in the tracts of neural systems that sub - serve a range of functions are required to evaluate more pervasive neuroprotective effects .
diffusion mri ( dmri ) and tractography enable measures of white matter tract integrity to be determined in specific tracts - of - interest ( tois ) ( basser et al . , 2000 ) .
, 1999 ) or probabilistic ( behrens et al . , 2007 ) approaches to identify the major pathways , with tracts initiated at seed points and typically constrained by operator - defined  waypoints
segmented pathways can then be analyzed as 3-dimensional regions - of - interest and water molecule diffusion parameters extracted .
neonatal dmri data present challenges to tractography algorithms because of smaller head size , resolution difficulties , wide anatomic variation , increased brain water content , and lack of reference atlas data from typically developing newborns .
consequently , neonatal studies have tended to focus on a relatively small number of tois , none has provided information about tract topology , and few have included control data . an alternative technique for segmenting tois
single seed point tractography is used to segment the same toi across a group of subjects by modeling how individual tracts compare with a predefined reference in terms of length and shape ( bastin et al . , 2010 ;
to achieve this , candidate seed points are automatically placed in a cubic neighborhood of native space voxels , with the tract that best matches the reference tract chosen as its representation in each subject ( bastin et al . , 2010 ; clayden et al . , 2006 ;
the output includes tract - averaged water diffusion tensor parameters , mean diffusivity ( d ) and fractional anisotropy ( fa ) , and the goodness - of - fit of the best - matched tract to the reference ( r ) .
the approach has been useful for mapping white matter change in normal development ( clayden et al . ,
2010 ) , and amyotrophic lateral sclerosis ( bastin et al . , 2013 ) .
pnt overcomes the bias inherent to user - defined waypoint placement , does not require thresholding of connectivity values , and is unique in providing information about tract topology . in this paper , we generated an atlas of reference tracts for eight tois using pnt to test the hypotheses that : 1 ) tract topology , and tract - averaged d and fa , are altered by preterm birth ; 2 ) tois are separable in the newborn brain based on water diffusion parameters ; and 3 ) pnt is sensitive to detecting perinatal neuroprotective treatment effects .
ethical approval was obtained from the national research ethics service ( south east scotland research ethics committee ) , and informed written parental consent was obtained .
preterm infants were recruited from the royal infirmary of edinburgh between july 2012 and september 2014 .
infants were eligible if they had a birth weight < 1500  g and/or were born at < 31 completed weeks ' postmenstrual age ( pma ) .
exclusion criteria for the study were infants with congenital infection , major chromosomal abnormalities , cystic periventricular leucomalacia , post - hemorrhagic ventricular dilatation and porencephalic cysts .
mri data from 24 healthy infants born at full term and 87 preterm infants ( mean pma at birth 29  weeks , range 2334  weeks ) were acquired at term equivalent age ( mean pma 39  weeks , range 3742  weeks ) ( table 1 ) .
infants were examined in natural sleep with pulse oximetry , temperature and electrocardiography data monitoring .
ear protection was used for each infant , comprising earplugs placed in the external ear and neonatal earmuffs ( minimuffs , natus medical inc .
a siemens magnetom verio 3  t mri clinical scanner ( siemens ag , healthcare sector , erlangen , germany ) and 12-channel phased - array head coil were used to acquire : t1-weighted mprage volume ( ~1  mm resolution ) , t2-weighted stir ( ~0.9  mm resolution ) , t2-weighted flair ( ~1  mm resolution ) , and dmri ( 11 t2- and 64 diffusion encoding direction ( b  =  750  s / mm ) single - shot spin - echo echo planar imaging ( epi ) volumes with 2  mm isotropic voxels ) data .
dmri data were pre - processed using fsl tools ( fmrib , oxford , uk ; http://www.fmrib.ox.ac.uk ) .
this included brain extraction , and removal of bulk infant motion and eddy current induced artifacts by registering the diffusion - weighted to the first t2-weighted epi volume for each subject .
bedpostx / probtrackx algorithm was run with its default parameters : 2-fiber model per voxel , 5000 probabilistic streamlines for each tract with a fixed separation distance of 0.5  mm between successive points to generate the underlying white matter connectivity data ( behrens et al .
eight reference tois were created using pnt in standard space from the tractography output produced from a training set of 20 term controls : genu and splenium of corpus callosum , left and right projections of the corticospinal tract ( cst ) , cingulum cingulate gyri ( ccg ) and inferior longitudinal fasciculi ( ilf ) .
candidate tracts were generated by seeding within a 7    7    7 neighborhood of native space voxels centered around a standard space seed point located within the center of the white matter structure - of - interest and transferred to the native space of each control .
these standard space seed points were defined using an age specific t2-weighted neonatal template ( http://www.brain-development.org ) ( kuklisova - murgasova et al . , 2011 ) .
then , an experienced observer ( da ) selected candidate tracts from each control subject that best matched the anatomical shape of the white matter structure - of - interest . using these visually - assessed  best - matching tracts ,
tract shape models were fitted to the tractography data using maximum likelihood ( clayden et al .
, 2007 ) , thereby capturing the shape and length variation amongst the controls ' tracts .
once the reference tracts and shape models had been created , pnt was re - run over a neighborhood of 3    3    3  voxels centered on the reference tract and transferred to native space for all participants ; the seed point that produced the best matching tract to the reference was then determined .
all the computational analysis was implemented in tractor ( http://www.tractor-mri.org.uk ) ( clayden et al . , 2011 ) .
two experienced raters ( da , meb ) visually assessed the best - matched tracts .
any subject with deviated , aberrant or truncated pathways that were not anatomically plausible representations of the toi were excluded from further analysis ( table 2 )
. the tractography masks of the best match tracts were applied to the d and fa volumes , which permitted tract - averaged values of these biomarkers to be determined for each toi in every subject . finally , the goodness - of - fit of the best match tract to the reference ( r ) was determined from the log - ratio between the matching likelihood of the chosen candidate tract and the matching likelihood of the reference tract under the model .
the more negative the r value , the less good the fit between the reference and best match tract .
the lateral ventricles were segmented in 59 out of 101 subjects for whom high resolution t1w and t2w structural data were available ( 10 controls and 49 preterms ) .
an atlas - based image segmentation approach was used that included lateral ventricles and choroid plexus , but excluded the third and fourth ventricle , cavum septum pellucidum and vergae ( serag et al . , 2012 ) .
this was done so that lateral ventricular size could be added as a co - variate to account for possible partial volume effects in the model testing independent effects of antenatal mgso4 on dmri parameters , and to investigate correlation between r and ventricular size .
the distributions of d , fa and r in each tract were assessed for normality using the shapiro 
whitney u test was used to compare r values between groups ; spearman 's rho was used to investigate correlations between r and pma at scan . because age at scan differed between the two cohorts , and water diffusion parameters are dynamic over this developmental period , glm univariate anova was used to investigate the effect of prematurity on these biomarkers in each toi with gestational age as a fixed factor and pma at scan as a covariate .
type 1 error was controlled using false discovery rate ( fdr ) ( p  <  0.05 ) . to investigate the effect of antenatal mgso4 exposure on tract - averaged d and fa , and r , independent of potential confounders
, we first examined the association between these biomarkers with the following factors : exposure to a complete course of antenatal steroids , exposure to antenatal mgso4 , gender and the presence of bronchopulmonary dysplasia ( bpd ) .
0.05 ) were included in a multivariate linear regression model that included pma at birth .
fa volumes were blurred in eight preterm infants and two controls due to motion ; these subjects were removed from further analysis leaving 79 preterm and 22 control infants in the analysis sample ( demographic shown in table 1 ) .
two preterm infants required laser treatment for retinopathy of prematurity and 1 had a grade 3 intraventricular hemorrhage ( ivh ) identified on cranial ultrasound . fig .
1 displays the reference tois that were identified reliably in the control training set in standard space . across both preterm and
control subjects , visual assessment of the individual segmented tracts indicated that pnt provided anatomically acceptable representations of the toi for the vast majority of tracts ( table 2 ) ; for the preterm cohort this ranged from 100% ( 79/79 ) for cst to 82% ( 65/79 ) for right ccg .
there was a significant difference in r between groups , with the preterm cohort having larger negative values , and hence greater topological difference from the reference compared to controls for all tois except the splenium ( table 3 ) .
2 illustrates the range of values for r observed in the preterm cohort and how they correspond to tract shape for genu . there were no significant correlations between age at scan and r , with the exception of left cst .
r correlated with ventricular volume for left ilf ( p  =  0.009 ) and left cst ( p  =  0.004 ) , but not with any other toi .
table 2 shows tract - averaged water diffusion parameter values for each toi for all participants . in bivariate analysis ,
tract - averaged d was higher in all tracts in preterm infants at term equivalent age compared with term controls , while fa was lower in all tracts except the right ccg .
after adjusting for pma at scan , tract - averaged d was increased in preterm infants at term equivalent age compared with controls in genu ( f  =  17.57 , p  <  0.001 ) , splenium ( f  =  19.22 , p  <  0.001 ) , left ilf ( f  =  8.92 , p  =  0.004 ) and right ilf ( f  =  8.36 , p  =  0.005 ) .
tract - averaged fa was reduced in the splenium ( f  =  7.16 , p  =  0.009 ) and left ilf ( f  =  6.73 ,
p  =  0.01 ) in preterm infants compared with controls after adjustment for age at scan .
3 displays a scatterplot of tract - averaged d versus fa values for the eight tois .
this figure indicates that cst , ccg , splenium and , to a lesser extent , ilf and genu are separable .
forty ( 51% ) of the preterm cohort had been exposed to antenatal mgso4 , and this was associated with lower d in splenium ( mean difference 0.089  mm / s , 95% ci 0.161 to 0.017 , p  =  0.016 ) . in multivariate analysis , antenatal mgso4 was associated with a modest but significant decrease in d in splenium independent of age at birth and bpd ( beta 115.0 , 95% ci 43.4186.5 , p  =  0.002 ) .
factors included in the model were : pma at birth because there was a difference between those who had [ mean pma 28  weeks ] and had not been exposed to antenatal mgso4 [ mean 29  weeks ] ; and bpd , because it influenced fa on univariate analysis ( mean difference 0.03 , 95% ci 0.05 to 0.005 , p  =  0.015 ) .
there was no effect of pma at scan , which did not differ between the two groups ( mean 39  weeks versus mean 40  weeks ) , or gender in univariate analysis , so these variables were not included .
ventricular volumes were available for a subset of 28 of the treated group and 21 of the untreated group .
there was no significant difference in ventricular volume between treated and untreated infants ( 8.4 versus 9.8  cm , p  =  0.11 ) , and in a subset analysis of infants where ventricular volume was entered into the model , antenatal mgso4 exposure retained significance ( beta 100.1 , 95% ci 28.8172.3 , p  =  0.007 ) .
these data suggest that the observed difference in d is unlikely to be attributable to the partial volume effects from csf .
we developed a single seed point tractography - based segmentation algorithm ( pnt ) that incorporates tract shape modeling for use in neonates , and have segmented reliably eight major fasciculi .
the algorithm is unique in providing a metric of tract topology , r , which measures the goodness - of - fit of the best matched tract with respect to an age specific tract template , and enables the extraction of tract - averaged water diffusion parameters including d and fa .
the advantages of pnt over region - of - interest and voxel - based methods for group - wise comparisons of d and fa are that it avoids the need for waypoint placement and setting of connectivity thresholds , and tracts are segmented in native space , which minimizes operator bias and takes account of individual differences in white matter structure .
the method uses connectivity data produced by a probabilistic tractography algorithm , fsl 's bedpostx / probtrackx , that can model multiple fiber populations and track through regions of reduced anisotropy , which has particular utility in neonates .
furthermore , by modeling the tracts of the whole study group with respect to reference tracts generated from an age specific training set , it is possible to perform group - wise analyses to study perinatal influences on r , and tract - averaged d and fa .
the measurement r , which indicates how the length and shape of the best - matched tract in each subject differs from reference tract topology , was significantly altered in preterm infants at term equivalent age compared with term controls in all tracts except splenium . to our knowledge
our data suggest that tract shape change occurs independently of d and fa , and is less influenced by age at scan than these measures .
therefore , r may add value beyond other biomarkers that are affected by age at scan , which can be challenging to standardize in neonatal studies .
a measure of tract shape is also particularly relevant given the wide anatomic variability of the brain at this stage of life , which is not captured in most tractography models .
further work is required to understand the functional correlates of r and whether it alters over time , but longitudinal studies of preterm infants suggest that alterations in structure and connectivity that are apparent in the neonatal period persist through early childhood into adulthood ( fischi - gomez et al . , 2014 ; nosarti et al . , 2014 ; pandit et al . , 2014 ) .
toga and thompson , 2005 ) , and the incorporation of geometric information about tracts has proven useful for studying heritability of brain integrity and connectivity inferred from dmri in adults ( jin et al . , 2011 ) .
we propose that r may offer a new tool for studying genetic contributions to brain structure in the newborn , which to date has been investigated using fa and morphometry but has not included shape analysis of major fasciculi ( boardman et al . , 2014 ) .
tract - averaged values of d were higher and fa values were lower in all eight tois in preterm infants at term equivalent age compared with controls in bivariate analysis .
after adjustment for pma at scan , which was necessary because both metrics change rapidly during perinatal brain development ( huppi et al . , 1998 ) , d was only increased in genu and splenium , while fa was reduced in the splenium and left ilf .
our findings are in broad agreement with previous region - of - interest ( counsell et al . , 2006 ;
, 2002 ; rose et al . , 2007 ; skiold et al . , 2010 ) , voxel - based ( gimenez et al . , 2008 ; thompson et al . , 2014 ) , and tractography approaches ( aeby et al . , 2009 ; anjari et al . , 2007
; thompson et al . , 2011 ) , which show that preterm infants at term equivalent age have higher d and lower fa in white matter compared with term controls , although the anatomic localization of these changes is inconsistent between studies and the role of confounding morbidities is uncertain .
the relationship between water diffusion biomarkers and white matter development is complex because the maturational processes of axonal fasciculation , pre - myelination ( proliferation of glial cell bodies , extension of oligodendroglial processes , increase in macromolecules and membrane density ) , and true myelination ( ensheathment of axons ) are all anisotropic ( beaulieu , 2002 ; nossin - manor et al . , 2013 ; zanin et al . ,
2011 ) , and the sequence of these processes is not uniform across neural systems . therefore , while dmri biomarkers provide sensitive measures of tissue microstructure , they are also non - specific .
we found that tracts were separable based on water diffusion parameters , with the greatest difference observed between cst , ccg and splenium .
this suggests that adult patterning of neural systems ( bastin et al . , 2010 ) is present very early in development , and before the onset of widespread myelination .
systematic review of 5 randomized controlled trials shows that antenatal mgso4 reduces the burden of cerebral palsy and other motor impairments in the offspring of women who deliver very preterm ( doyle et al . , 2007 ) , but there is some doubt about long term benefit and none of the studies were designed to assess function in other domains as a primary outcome ( chollat et al . , 2014 ; doyle et al . , 2014 ) .
magnesium is thought to act on a number of cellular targets and processes in preterm brain injury , which are not restricted to motor systems , so it is plausible that tissue effects might be apparent in other neural systems .
we found a modest but significant reduction in d in the splenium of preterm infants who had been exposed to antenatal mgso4 compared with untreated infants , after adjustment for relevant confounders .
the splenium connects parietal , temporal and visual cortices , so these data indicate that antenatal mgso4 exposure has beneficial tissue effects that extend beyond motor systems .
detailed evaluation across all developmental domains of infants exposed to antenatal mgso4 is needed to understand fully its treatment effects .
finally , our data suggest that tract - averaged water diffusion parameters may provide useful biomarkers to complement voxel - wise approaches ( such as tract - based spatial statistics ) for evaluating neuroprotective strategies in the newborn ( o'gorman et al . , 2015 ; porter et al . , 2010
first , we were unable to identify uncinate and arcuate fasciculi in the training set although these tracts can be segmented from adult data using pnt ( clayden et al . , 2007 ) ; this is likely due to lower resolution inherent to neonatal dmri or relative under - development of these tracts at term equivalent age .
second , the success of pnt varied from 100% for cst to 82% for right ccg in the preterm group , which may be explained by differential susceptibility to partial volume effects for different tracts .
a further potential limitation relates to multiple testing : eight simultaneous tests were carried out for the d and fa data respectively .
bonferroni correction is inappropriate here because tract - averaged fa and d values are correlated across the brain ( penke et al . , 2010 ) , so we chose to control the effects of multiple testing with fdr at a threshold of 0.05 . finally , the choice of reference template is a potential source of bias : we chose a reference based on healthy controls born at term because it provided the most biologically 
further work would be required to determine whether a reference template drawn from a representative sample of the entire study group influences the results .
we have shown that pnt , an automated single seed point tractography method , segments tois with a high degree of accuracy in the newborn brain .
it is unique in providing information about tract topology , and it enables calculation of tract - averaged dmri biomarkers .
tract topology is a marker of preterm birth that is relatively robust to age at scan .
we found that discrete neural systems can be separated by water diffusion parameters , reflecting specific microstructural properties of tracts that are apparent in early life , and that tract - averaged dmri biomarkers provide a sensitive metric for evaluating risk factors for preterm brain injury and neuroprotective treatment effects .
the approach may have utility for investigating early life determinants of long - term neurologic and psychiatric disorders .
 preterm birth is associated with altered connectivity of neural circuits . 
 we developed a tract segmentation method that provides measures of tract shape and integrity ( probabilistic neighborhood tractography , pnt ) from diffusion mri ( dmri ) data to test the hypotheses : 1 ) preterm birth is associated with alterations in tract topology ( r ) , and tract - averaged mean diffusivity ( d ) and fractional anisotropy ( fa ) ; 2 ) neural systems are separable based on tract - averaged dmri parameters ; and 3 ) pnt can detect neuroprotective treatment effects.dmri data were collected from 87 preterm infants ( mean gestational age 29 + 1  weeks , range 23 + 2 34 + 6 ) at term equivalent age and 24 controls ( mean gestational age 39 + 6  weeks ) . 
 pnt was used to segment eight major fasciculi , characterize topology , and extract tract - averaged d and fa.tract topology was altered by preterm birth in all tracts except the splenium ( p  <  0.05 , false discovery rate [ fdr ] corrected ) . after adjustment for age at scan , tract - averaged d was increased in the genu and splenium , right corticospinal tract ( cst ) and the left and right inferior longitudinal fasciculi ( ilf ) in preterm infants compared with controls ( p  <  0.05 , fdr ) , while tract - averaged fa was decreased in the splenium and left ilf ( p  <  0.05 , fdr ) . 
 specific fasciculi were separable based on tract - averaged d and fa values . 
 there was a modest decrease in tract - averaged d in the splenium of preterm infants who had been exposed to antenatal mgso4 for neuroprotection ( p  =  0.002).tract topology is a biomarker of preterm brain injury . 
 the data provide proof of concept that tract - averaged dmri parameters have utility for evaluating tissue effects of perinatal neuroprotective strategies . 
Introduction
Methods and materials
Results
Discussion
Conclusions