Alcohol 36 (2005) 151–160

Preliminary evidence that prenatal alcohol damage may be visible in

averaged ultrasound images of the neonatal human corpus callosum
Fred L. Booksteina,b,c,*, Paul D. Connora, Kristi D. Covella, Helen M. Barra,
Christine A. Gleasond, Raymond W. Szee,f, Jenny A. McBroomf, Ann P. Streissgutha
a
Fetal Alcohol and Drug Unit, Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, WA 98195, USA
b
Department of Statistics, University of Washington, Seattle, WA 98195, USA
c
Department of Anthropology, University of Vienna, Vienna A1091, Austria
d
Division of Neonatology, Department of Pediatrics, University of Washington, Seattle, WA 98195, USA
e
Department of Radiology, University of Washington, Seattle, WA 98195, USA
f
Children’s Hospital and Regional Medical Center, Seattle, WA 98105, USA
Received 11 April 2005; received in revised form 1 July 2005; accepted 22 July 2005

Abstract
Brain damage consequent to prenatal alcohol exposure can be detected by measurements of the corpus callosum in the midline magnetic
resonance (MR) brain image in adolescents and adults. The present article extends this finding into the neonatal period, when the power of
detection to ameliorate the quality of the child’s future life is greatest. The midline corpus callosum of the very young infant can be located
reliably in multiple frames of clinical transfontanelle ultrasound. We studied a sample of 18 children aged 17 weeks or less, 7 of whom were
exposed to high levels of alcohol prenatally and 11 of whom were not exposed or only minimally exposed. The midline callosum of each
child was imaged up to 50 times by a standard clinical device, and coplanar subsets of these series were averaged with reference to fiducial
image structures. On each average image four semilandmark points were set and their configuration quantified by standard landmark meth-
ods. The angle between the terminal bulb of splenium and the long axis of the callosal outline classifies four of the seven exposed infants as
different from all 11 of the unexposed infants. This simple angle measurement upon averaged ultrasound images of the human neonatal
midline corpus callosum, perhaps a version of the long-sought ‘‘biomarker of prenatal alcohol damage,’’ may be able to discriminate baby
brains affected by prenatal alcohol exposure from those that were unaffected. Ó 2005 Elsevier Inc. All rights reserved.
Keywords: Fetal Alcohol Spectrum Disorders; Corpus callosum; Neonatal ultrasound; Unwarped image averaging; Shape coordinates; Splenium

1. Introduction amply confirmed that the embryonic tissue most sensitive to

alcohol’s effects is the developing brain and have demon-
The announcement of fetal alcohol syndrome (FAS) in
strated several mechanisms of this damage: disruption of
1973 (Jones & Smith, 1973; Jones et al., 1973) was based
early astrocyte, glial, and neuronal migration (Guerri &
on examinations of 11 Seattle children who all had the
Renau-Piqueras, 1997; Phillips, 1994); selective loss of hip-
same unusual facial features and whose mothers had all pocampal pyramidal cells and cerebellar Purkinje cells
abused alcohol during that pregnancy. In the three decades (Bonthius & West, 1990); alcohol-induced neuroapoptosis
since then, this initial publication has ramified in many dif-
during synaptogenesis (Ikonomidou et al., 2000); disruption
ferent directions (Stratton et al., 1996; Streissguth, 1997). A
of neurotransmitter systems and neurotrophic excitatory
rich range of clinical studies, some retrieved from the pre-
activity (Olney et al., 2000); and dysregulation of cellular
1973 literature and others published since then, make it
adhesion (Charness et al., 1994).
clear that this disease is found in every region of the world
At the same time, as the typical profile of neuropsycho-
and in every human race or ethnicity. Animal models have
logical deficit in human patients with prenatal alcohol dam-
age has been explored in great detail (Mattson & Riley,
1998), along with its patterns of variation (Bookstein
* Corresponding author. Fetal Alcohol and Drug Unit, 180 Nickerson
Street, Suite 309, Seattle, WA 98109, USA. Tel.: 11-206-543-7155; fax:
et al., 2002b), it has become clear that the original facial
11-206-685-2903. characterization of FAS underrepresents the range of pa-
E-mail address: flb@stat.washington.edu (F.L. Bookstein). tients damaged by prenatal exposure to alcohol. For
0741-8329/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved.
doi: 10.1016/j.alcohol.2005.07.007
152 F.L. Bookstein et al. / Alcohol 36 (2005) 151–160
instance, it is now known that persons exposed to high lev-
els of alcohol before birth show similar average neurobeha-
vioral deficits regardless of the presence of the classic facial
stigmata (Mattson et al., 1998) and that within this general
pool of patients the extent of brain damage is not tightly
controlled by the presence or absence of the facial stigmata
(Bookstein et al., 2002a). Recently, by analogy with other
wide-spectrum birth defects, a term was coined, Fetal Alco-
hol Spectrum Disorders (FASD), that embraced the classic
FAS face as well as other classes of patients (fetal alcohol
effects [FAE], alcohol-related neurodevelopmental disor-
der, etc.) defined in terms of central nervous system
(CNS) effects (NOFAS, 2004; Streissguth & O’Malley,
2000). Estimates of prevalence of this broader condition
ranged up to about 0.9% in a population-based study with
blinded infant examination (Sampson et al., 1997).
Although the original CNS characterizations of FAS
used mainly global measurements, such as head circumfer-
ence or full-scale IQ, it has since become clear that some
regions of the developing brain are more sensitive and spe-
cific than others (in the statistical sense) to these alcohol
effects. Riley et al. (1995) noted the disproportionate
reduction of corpus callosum size, on the average, in FASD
patients, and Swayze et al. (1997) confirmed this in another
series. More recently, our own group has found a strong sta-
tistical signal for neuroanatomical damage in FASD, strong
enough for effective classification of persons with FASD
vis-à-vis age-comparable unexposed subjects, in the details
of form of the corpus callosum outline in the midline
(Bookstein et al., 2001, 2002a, 2002b). This finding, which
pertains to adolescents and adults of both sexes, emphasizes
variation of the callosal midline shape in any of a wide
range of possible dimensions of dysmorphology: hypervari-
ability of almost every possible pattern measure. Detection
is by quadratic discrimination in many dimensions, not by
any specific linear combination.
Yet in this burgeoning literature one theme has been left
oddly fallow: the possibility of diagnosis right at the time of
birth, as in several of those initial 11 patients (Jones &
Smith, 1973; Jones et al., 1973). We know from studies
of acquired ‘‘secondary disabilities’’ (Streissguth et al.,
2004) that the earlier that FASD is diagnosed, the better
the prognosis for an accommodation of society to the pa-
tientdfor instance, improved prospects for amelioration
of those secondary disabilitiesdonce the patient has
reached his or her own limits of accommodation. Inasmuch
as most people with FASD are not raised by their biological
mothers (Streissguth et al., 1991a, 2004), early identifica-
tion would better inform the fostering and adoption of these
children and would help babies with brain damage from al-
cohol to get into the existing early intervention programs
that would best serve them (Whitney et al., 2004). Further-
more, earlier diagnosis of babies would also help mothers
with alcohol problems to get into appropriate programs to
prevent future alcohol-affected pregnancies (Grant et al.,
2003), and would enable better longitudinal studies of the
variability of this spectrum disorder and the relative effica-
cy of proposed interventions.
It is therefore appropriate to return to the pediatric
theme, via studies that detect the alcohol-damaged off-
spring as early as possible. Wass et al. (2001), in a study
of 155 fetuses imaged by ultrasound, noted substantial cor-
relations between a categorization of alcohol exposure and
frontal lobe length (from cavum pellucidum to calvarium)
adjusted over a range of gestational ages from 15 to 36
weeks, and other studies conducted as part of the same Col-
laborative Initiative on FASD that supported the research
reported in this article are likewise attempting to character-
ize the effect of prenatal alcohol exposure on the brain be-
fore birth. To fill the gap between the fetal period and
postinfancy life, it would be very useful to pursue protocols
for the detection of fetal alcohol damage at the earliest pos-
sible postnatal age. In this article we report a very promis-
ing finding, a possible neuroanatomical marker of fetal
alcohol damage, in a sample of children imaged in the first
3 months of postnatal life by a relatively inexpensive and
widely available imaging modality, the pediatric ultrasound
machine. Subsequent sections of this article set out the sam-
pling design of this study, image acquisition and measure-
ment protocols, and the nature of the neuroanatomical
marker signal that we believe to have been uncovered. A
concluding discussion sketches implications of this surpris-
ing finding for the field of fetal alcohol research and for pe-
diatric public health more generally.
2. Materials and methods
2.1. Samples
We referred 20 infants to the Radiology Department of
Children’s Hospital and Regional Medical Center (after ap-
proval by the Institutional Review Board there) for trans-
fontanelle ultrasound imaging of the midline corpus
callosum. Babies of more heavily drinking mothers were
recruited from programs serving high-risk mothers and
their babies. Babies of infrequent drinkers and abstainers
were a sample of convenience recruited from a range of lo-
cal pools: a University of Washington research Web site for
‘‘volunteers,’’ a local prenatal clinic that provides midwife
services, response to posters at the University hospital, re-
sponse to handouts at parent education classes at the Uni-
versity, and referrals from our own staff members or from
other unexposed subjects. Screening for eligibility was
done using the Hospital Screening Questionnaire (HSQ)
(Streissguth et al., 1991b). This is a one-page questionnaire
about alcohol and drug use during two time periods: during
pregnancy and in the month or so before the mother was
pregnant or knew she was pregnant. The HSQ was scored
according to the Binge Alcohol Rating Criteria (BARC)
(Barr & Streissguth, 2001). A woman who was BARC1
(read ‘‘bark positive’’) or almost BARC1 according to
the HSQ was given the Pregnancy Drinking Calendar
 F.L. Bookstein et al. / Alcohol 36 (2005) 151–160 153

(PDC), which asks for more detail. The BARC score is
a weighted sum of the monthly frequencies of three to four
drinks per occasion and five or more drinks per occasion as
reported for either time period. Information from the PDC
was useful in some exposed cases to determine whether
they met or fell short of the published BARC threshold.
For the exposed cases in the present sample, the BARC1
code was equivalent to a frequency of four or more binges
(five or more drinks on an occasion) per month in one or
both of the time periods (see Table 1).
Babies of mothers who were BARC1 according to ei-
ther of these instruments were eligible for the study. Moth-
ers in the comparison group reported no alcohol intake or
almost no alcohol intake during either time period on either
the HSQ or the PDC. Babies who were less than 33 weeks
gestation at birth or older than 4 months of age at the ear-
liest feasible time of postnatal ultrasound or whose mothers
reported taking any street drugs (except marijuana or co-
caine) or any drug known to be teratogenic were excluded.
We initially thought that imaging needed to be completed
by age about 17 weeks because thereafter we expected that
the fontanelle would be insufficiently patent for a good ul-
trasound view of the midline callosum. Apart from these
age considerations, there was no selection for any charac-
teristic of the infant.
2.2. Images
After informed consent was obtained from mothers, all
20 infants were imaged by transfontanelle ultrasound using
a standard modern clinical scanner at Children’s Hospital
(an HDL ultrasound scanner with a C8-5 pediatric cephalic
transducer and cine memory). Gestational age ranged from
33 to 42.5 weeks, and imaging was done from 5 to 16.6
weeks after birth. The ultrasonographer was directed to
hold the transducer fixed over the child’s anterior fontanelle
and, by dynamically oscillating the image plane (while ac-
commodating the simultaneous squirming of the unsedated
baby), to capture up to 50 freeze-frames that were as close
to the midsagittal section as possible, as judged by the im-
precise image of the cerebral aqueduct together with char-
acteristic sulci of the parasagittal cortex, and that included
the full length of the midline callosum (our intended target
of measurement). These frames were black–white reversed
and exported for analysis by an existing module of the
Edgewarp program package (Bookstein & Green, 1994).
Until the individual image processing had been completed
for all 20 subjects, the analyst (FLB) was blinded as to sam-
ple totals of exposed and unexposed and also, of course, to
the exposure status of each subject.
2.3. Unwarped image averaging
It is notoriously difficult to quantify shape in ultra-
sound imagery. In studies of moving structures, such as
the heart, frame-to-frame correlations permit analysis of
motion separate from feature analyses of individual
frames of the movie. For quantitative studies of form,
we need a somewhat analogous toolda serial registration
tooldthat builds measurable structures out of hints in the

Table 1
Ages, exposures, and angle at splenium for 18 infant subjects
Age at Other Angle at
Subject number, ultrasound reported splenium
gender Binges Age at birth (weeks) Birthweight (weeks) exposures (degrees)
Heavily alcohol exposed (N 5 7)
11F (12,3) 40.0 8.10 13.1 (none) 63
8M (3,4) 41.0 8.0 8.4 cig, mar, coc 64
5F (2,4) 33.0 NA 11.3 cig, mar, coc 82
2M (30,6) 40.0 8.14 16.1 mar 92
9M (10,NA) 39.0 6.11 7.4 mar, coc 94
10F (4,2) 42.0 6.14 7.9 cig, mar 106
4F (30,30) 39.0 NA 16.4 cig, mar, coc 118
Comparison group (N 5 11)
22F 42.5 6.13 4.9 cig, mar 51
16M 35.0 5.7 15.4 (none) 58
23F 39.0 6.3 7.1 cig 62
15F 35.0 5.8 15.1 (none) 62
6M 39.0 10.8 16.4 (none) 64
13F 39.0 7.5 16.4 (none) 65
12F 41.0 7.12 14.3 (none) 69
14F 42.0 8.14 12.3 (none) 71
20F 41.0 7.8 11.0 (none) 73
7F 37.0 6.10 11.9 (none) 73
3F 39.0 8.6 16.6 (none) 83
Note: Ordering is by angle at splenium within alcohol exposure group. Binges 5 monthly frequency of binges of five or more drinks per occasion, before
and during pregnancy. All binge scores in the comparison group are 0. NA 5 not available, birthweight 5 pounds and ounces, Cig 5 cigarettes, Mar 5 mar-
ijuana, and coc 5 cocaine. M, F: male, female.
154 F.L. Bookstein et al. / Alcohol 36 (2005) 151–160

separate frames. For this purpose we have tested a system of splenium and the segment from tip of genu to tip of sple-
of unwarped image averaging that proceeds in the follow- nium (see Fig. 1). Individual scalar measurements such as
ing way. distances and angles are a risky way of recording differen-
For each infant, the analyst selected a subset of images, ces in configurations; it is better to locate suitable land-
numbering from 6 to 30, that indeed captured the complete marks and semilandmarks (Bookstein, 1991) on the form.
length of the midline callosum and that appeared to show For a small sample of forms, it is best to use a similarly
nearly the same plane of image sectiondthe same general small selection of shape coordinates. As shown in Fig. 1,
callosal outline shape, with the same characteristic identify- a mere four points (eight shape coordinates’ worth) were lo-
ing markings. The fraction of frames accepted for analysis cated in each averaged unwarped image from the series of
varied from 30% to 75%, averaging 40% for the unexposed 18. One landmark was placed right at the anterior tip of
infants and 55% for the exposed infants (difference nonsig- genu and another at the posteroinferior tip of splenium.
nificant by Wilcoxon test). On each frame of the selected Separately, additional semilandmarks (‘‘sliding land-
subset, fiducial points (reference points specific to the indi- marks,’’ Bookstein, 1997) were placed at the midpoint of
vidual) were placed at the presumptively fixed location of the arch (the point on the upper margin of the arch perpen-
the transducer (the center of the visible fan beam) and at vi- dicularly above the midpoint of the segment from genu tip
sually characteristic points in the vicinity of genu and of to splenium tip) and, purely as an attempt to record the ori-
splenium. (Thus, these points are not landmarks in the mor- entation of the splenium, at the splenium intercept point on
phometric sensedthey are in biologically different loca- or near the upper border of the isthmus where a linear ap-
tions from brain to brain, although in the same locations proximation to the medial axis of the splenium (fitted by
for successive images from a single brain.) The resulting eye) would intersect it.
stack of fiducially marked images was processed by the av- These four-point configurations were analyzed by stan-
eraged image unwarping method (Bookstein, 1999), which, dard two-point shape coordinate methods (Bookstein,
for the simplest fiducial configuration here (three points 1991).
only), reduces to (1) an operator-determined affine transfor- When the exposure data were added in, we found that
mation (translation, rotation, and shear) applied to each im- there were 8 exposed and 12 unexposed children in our
age in turn to superimpose its three points over their average
positions, followed by (2) a pixel-by-pixel averaging of the
image information after all transformations have been ap-
plied. (Thus, in the sample images below, the machine’s
standard text block, originally in the same location on the
videoframe, is shifted into various positions, close together
if the text was near the transducer and arbitrarily far apart if
in the margins of the image for a baby who was squirming
actively during the procedure.) These averaged images have
not been edited or tidied up, and so the limits of the original
freeze-frames are clear around the margins. For one baby,
subject 10, the scale of the fan beam upon the video capture
device was apparently reset during the scan; this does not
affect the subsequent analysis of that baby.
The two groups were not matched and the study was not
balanced when the cutoff date arrived. Of the 20 infants
scanned, two had fewer than six coplanar and complete ul-
trasound frames to be processed by this method. (One of
these two was the very first baby imaged, when fewer im-
ages were obtained; the other was the oldest baby, possibly
with too small a fontanelle.) The protocol has now been ad-
justed to emphasize scanning during the first 3 months (13
weeks) of postnatal life.

2.4. Quantification
During data preparation, before exposure statuses were
revealed, we noticed that four of the averaged images were
strikingly abnormal: subjects 4, 10, 8, and 22. For three of
these, the abnormality could be flagged by a simple scalar
measurement, the angle between the long axis of the body
Fig. 1. Four-point representation of arch form, unwarped averaged im-
ages. Clockwise around arch from left: tip of genu (a landmark); top of
arch (a semilandmark taken exactly halfway between tip of genu and tip
of splenium); ‘‘angle-of-splenium point’’ (a semilandmark taken where
the long axis of splenium intersects the upper arch margindsee text);
tip of splenium (a landmark). The exemplar here is baby 10 (Fig. 2, middle
right). Inset: the location of ‘‘the angle at splenium.’’
 F.L. Bookstein et al. / Alcohol 36 (2005) 151–160 155

sample. Of the two excluded infants, one turned out to be 3. Results

from each group. Final group sizes are thus 7 exposed ver-
sus 11 unexposed infants. Now the four-point landmark/
semilandmark configurations could be analyzed by group.
The four points of each averaged image, like any other
configuration of landmarks and semilandmarks, were
themselves averaged within groups, and each infant’s aver-
aged image was unwarped to its own group’s (semi)land-
mark average configuration, and averaged there within
group.
3.1. Preliminary observations of single subjects
Some of our averaged images looked perfectly normal,
like the callosal outlines in the neonatal textbooks (Haller
et al., 1998): for instance, baby 12 (Fig. 2, upper left).
Others looked abnormal, in particular the two that had
the oddest-looking splenia (Fig. 2, middle row); these two
were from the group of seven subjects who had in fact
had high prenatal alcohol exposure. It was reasonable to

Fig. 2. Six interesting averaged unwarped images. Upper leftdbaby 12, very similar to textbook images of the ‘‘normal.’’ Upper rightdbaby 3, the unex-
posed infant with the highest angle of splenium. Middle rowdthe two babies of highest apparent splenium angle (4 and 10), both from the exposed group.
Bottom rowdthe two babies of hypoplastic splenium, one exposed (8, left) and one unexposed (22, right).
156 F.L. Bookstein et al. / Alcohol 36 (2005) 151–160

expect, then, that a quantification of the form of the sple- apparently has a sensitivity of 4/7 vis-à-vis exposure (four
nium might be able to tap some information about the effect infants are detected out of seven exposed) and a specificity
of the exposure. of 11/11 (no unexposed are misdetected out of 11).

3.3. Further averaged unwarped images

3.2. Two-point shape coordinates
As Fig. 3 shows, there are substantial differences be-
tween our two groups in the distribution of the coordinates
of the splenium intercept point. The meaningful coordinate
(the one implied by our characterization of the two images
in the middle row of Fig. 2 as ‘‘abnormal’’ prior to breaking
the exposure codes) is the horizontal coordinate in the en-
largement at right (subject numbers not in parentheses are
exposed subjects). The coordinates for the seven exposed
infants are surprisingly separate from the others: The four
highest values of the angle of the splenium medial axis to
the genu-splenium baseline of this construction are seen
in the alcohol-affected group, even though it comprises less
than 40% of the full sample of 18. A t test of the coordinate
in this direction returns a p value of less than .0012 for the
mean comparison. But, also, the variance of this relative
orientation in the exposed group is nearly 4 times that in
the unexposed ( p 5 .04 by ordinary F test), confirming
the general finding of hypervariability of callosal form that
we reported in our adolescent–adult samples (Bookstein
et al., 2002a). Now that the variances are shown unequal,
the t test might be replaced by a distribution-free version.
It is quite reassuring that a permutation test of the mean dif-
ference between the groups yields a p value of 11/10,000
(two tailed) using 10,000 permutations.
For the discussion below, it is useful to set out a specific
criterion of ‘‘detection’’ from these measures. The intuitive-
ly simple threshold of 90  dan angle at splenium that is ob-
tuse instead of acutedclassifies four of these seven exposed
infants without misclassifying a single unexposed infant. If
that angle is used as the criterion measurement, it
As Fig. 4 displays, there is a clear difference in the four-
point unwarped averaging of the landmark/semilandmark
configurations shown to be statistically significantly differ-
ent in this way. Of course these images are wildly out of
register except in the vicinity of the callosal arch itself; they
are not themselves intended to be of any analytic use. The
average angles differ by around 20  between left and right
panels.
Various data useful for the following discussiondgesta-
tional age at birth and at scanning, birthweight, the dispos-
itive components of the BARC score, other exposures, and
splenium angledare shown in Table 1.
4. Discussion
We may précis the findings here in four summary
assertions:
 The midline corpus callosum of the very young infant
(birth through 3 months) can be imaged reliably via
careful postprocessing of standard clinical ultrasound
images by averaged unwarped imaging.
 A substantial fraction of exposed infants show a poten-
tially diagnostic abnormality of the splenium in these
averaged unwarped images.
 Quantification of these averages seems straightfor-
ward using otherwise standard landmark and semi-
landmark methods, once the images themselves are
produced.
 The actual classification protocol associated with this
quantification finds an angular abnormality in four of

Fig. 3. Two-point shape coordinates of top of arch and angle of splenium to a genu-splenium baseline. Leftdall shape coordinates; rightdenlargement of
the distribution of the angle-of-splenium semilandmark. Shape coordinate pairs for babies in the exposed group are marked by subject number (see Table 1);
shape coordinates for the unexposed group are set with solid dots except for the possibly hypoplastic case 22 (Fig. 2, lower right), which is also noted here by
number in parentheses. The angle measurement reported in the text is with respect to the horizontal in this plot, as shown.
 F.L. Bookstein et al. / Alcohol 36 (2005) 151–160
Fig. 4. Further unwarped averaged images from the four-point representation of Figs. 1 and 3. Leftdaveraged both within and between subjects, for the 11
unexposed babies; rightdfor the seven exposed babies. The shape difference in the vicinity of splenium, which is visually obvious, is confirmed by t test,
along with a separate test of the hypervariance of the angle described in the text.
the seven infants known exposed to high levels of al-
cohol. The quantification, then, may be a detection
tool, not merely a group characterization.
The general level of accuracy of the corresponding de-
tection protocols (sensitivity 4/7, specificity 11/11) is com-
parable to that reported for our adolescent–adult sample
as imaged by magnetic resonance (MR) (Bookstein et al.,
2002a), and seems promising enough to flag these children
of abnormal splenium–callosum relationship as at increased
risk of neuropsychological or neurobehavioral deficits
throughout later development. The focus of the measure-
ment on the midline callosum is concordant with a qualita-
tive literature dealing with such concerns as callosal
agenesis (Bookstein et al., 2004; Jeret et al., 1986), with di-
verse earlier quantifications (Bookstein et al., 2001, 2002a;
Riley et al., 1995), and with at least one of the mechanisms,
disrupted neuronal migration, by which the deleterious con-
sequences of alcohol exposure are effected. It is thus not
unreasonable to hope that neonatal callosal dysmorphology
might be the crucial mediating variable between exposure
and detection of affected offspring by a variety of measure-
ments later in postnatal life. The hypervariability of callosal
form that is significant even in this small study confirms the
finding of our larger study of adolescents and adults and
holds out further promise for detection and for prediction
of differential profiles of fetal alcohol neurobehavioral def-
icit (Bookstein et al., 2002b) by measurable aspects of the
same neonatal callosal image. (Just as a mean difference
detected statistically can be converted into a tentative detec-
tion procedure by the standard linear discriminant function,
so a difference in variances detected statistically can be
converted into a tentative detection procedure by a quadratic
discriminant analysis. Recently, Sampson et al. (2005)
157
explicitly demonstrated both detection and profile predic-
tion by exploiting the combination of mean shift and hyper-
variance of callosal outlines in the alcohol-damaged via
formal quadratic discriminant analysis in a full multivariate
context.)
4.1. Limitations
The image processing here was manual, and would sure-
ly be more reliable (especially in screening contexts) were
it automated in respect of control point assignment (Meyer
et al., 1999) and midplane determination in these incom-
pletely realized images. (The effect of automation would
be not so much to change the geometry of these avera-
gesdfor instance, the shears entailed in the three-point reg-
istration should average out when the fiducial point
configurations themselves are averaged, which is the case
in our algorithmdas to sharpen the averaged image for
more precise assessment of callosal contours and curva-
tures.) There is an urgent need for formal norms of the nor-
mal neonatal callosum, including the effects of such
covariates as ethnicity, birthweight, and age, to substitute
for the artificial and inefficient inclusion of unexposed new-
borns in the screening context sketched above. The present
sample is far too small to check for other exposures as co-
variates, but when larger samples are available such neces-
sary further verifications become practicable. A larger
sample would justify, too, a greater concern for sampling
frame and other aspects of generalizability. In larger sam-
ples the somewhat arbitrary threshold of 90  suggested
above could likewise be replaced by an empirical optimum
(Swets & Pickett, 1982), and a more extensive quantifica-
tion might be assayed that involved, for instance, additional
158 F.L. Bookstein et al. / Alcohol 36 (2005) 151–160
measures at Rostrum (which is not often complete in this
series of images) or values of width and curvature function-
als along the arch as a whole (Bookstein, 1991). A design
such as that of Wass et al. (2001), concerning a larger sam-
ple with ample attention to covariates and confounds,
would clearly be worth considering as long as the strengths
of this simple study (explicit image averaging, shape coor-
dinate analysis) also persevere.
4.2. Modifications for screening: application to pediatric
public health
The pilot study here was a planned comparison of infants
from two separate groups known in advance to have highly
disparate levels of exposure to alcohol. For routine use in
pediatric public health, we recommend an alternate mode
of sample accrual, by way of maternal questionnaire (Barr
& Streissguth, 2001). Answers to two simple questions in
a brief and innocuous questionnaire can assess the infant’s
exposure to risk levels of drinking with sufficient accuracy
to justify referral of the high-exposed, together with a subset
of unexposed, for routine imaging. The ultrasound machine
itself is standard in high-risk neonatal wards, and the image
processing is not tedious and runs on free software (once
the freeze-frames have been offloaded from the clinical ma-
chine’s own video memory). This study was a small one. A
confirming study is currently underway in Vienna, Austria,
to validate this claimed yield of positive callosal findings
from children of women reporting high prenatal alcohol ex-
posure versus those not reporting that exposure. In any
screening application, it is crucial that the image be not on-
ly clinically examined but actually measured. Our neurora-
diologist flagged none of these cases, from either exposure
group, as showing clinically significant problems on the ba-
sis of these image sequences. A comparable absence of sen-
sitivity (2/117) characterized the clinical reading of the MR
brain images in adolescents and adults from which the find-
ing of callosal hypervariability initially derived (Bookstein
et al., 2002a).
4.3. Concomitant drug exposures
As Table 1 indicates, mothers of the babies exposed to
high levels of alcohol in this study report other exposures
as well. Within the constraints of this pilot study, it proved
nearly impossible to recruit women by referral whose off-
spring were exposed solely to alcohol. Four of the alco-
hol-exposed babies were born to smokers, versus only
two in the unexposed group; auxiliary data not otherwise
tabulated here indicate that the median daily number of cig-
arettes smoked was less than a pack a day in both these sub-
samples. Six of the seven alcohol-exposed babies were
exposed to marijuana as well, versus one in the unexposed
group. But dosage by marijuana is much less frequent in the
exposed group than dosage by alcohol (median, 33 drinks
per month prior to awareness of pregnancy, vs. 3.5 uses
of marijuana per month), making any homogeneity of effect
quite unlikely across this group. And four of the alcohol-
exposed babies were exposed to cocaine, versus none in the
unexposed group.
Marijuana use being so nearly coextensive with high al-
cohol exposure in this sample, the possibility that the group
difference here is confounded by marijuana exposure is
real. However, there is no reported literature of effects of
marijuana or of cocaine upon the form of the human corpus
callosum, let alone effects similar in their splenium-specific
geometry to those detected here. Likewise, Wass et al.
(2001) found no significant confounding by marijuana or
cocaine upon the regionally specific measurement (frontal
lobe length) that is the focus of the alcohol exposure effect
reported there, although they noted an interaction of alcohol
with nicotine. From the point of view of public health,
when alcohol abuse is coupled to polydrug abuse (as it is
in the mothers of these exposed children) and in the ab-
sence of specific brain damage consequent to prenatal mar-
ijuana exposure, it is the signal of geometrically specific
brain damage here that is more important for neonatal
and pediatric neurology. If polydrug exposure is indeed
the culprit here, that fact could be unearthed by a different
sampling design, with more numerous cases, better stratifi-
cation of other drug exposures over alcohol exposure, and
considerably greater care in the assessment of those other
exposures.
4.4. The hypoplastic neonatal callosum
A hypoplastic callosum is not an uncommon finding in
children and adult FASD samples (Riley et al., 1995;
Swayze et al., 1997). Case 8 of this study (Fig. 2, lower left)
may be an analogous example in this neonatal data set. The
Boolean combination of obtuse splenium angle or apparent
hypoplasia (here, not a quantification, just a visual appear-
ance) flags five of the seven exposed babies in this study,
along with one false positive (baby 22) who would, of
course, not have been scanned were the study running in
dose screening mode. Because not all exposed children
are in fact affected, this might be the truly affected five
of the exposed seven. If we slip the threshold of splenium
angle from 90  to 80  or so, in combination with hypopla-
sia, we flag six of the seven known exposed, now with two
false positives: a sensitivity of 6/7 and a specificity of 9/11
for detection of exposure. As shown in Table 1, the two hy-
poplastic cases have in common an exposure to marijuana,
and the hypoplastic callosum unexposed to alcohol is the
only such case exposed to marijuana. Although only anec-
dotal in a sample so small, this hint about an alternative
dysmorphological pathway or mechanism may merit fur-
ther investigation.
4.5. Follow-up
We are eager to follow these children up through an age
at which we can confirm the FAS diagnosis per se by facial
 F.L. Bookstein et al. / Alcohol 36 (2005) 151–160
measurement or else confirm the brain damage either by
MR imaging, if a clinical referral can be generated some-
how, or by measures of neuropsychological or neurobeha-
vioral deficits. In a group of 120 adolescents and adults
balanced by sex and by diagnostic category (FAS or FAE)
within FASD, Sampson et al. (2005) find the strongest cor-
relates of midline callosal form at later ages to be a wide
range of IQ-loaded measures together with some assess-
ments of executive function. The infant equivalents of
these, such as A-not-B, might be attractive candidates for
confirming neuropsychological scores in these infants as
well, along with the currently promising measures of alco-
hol-related brain damage in other areas such as eyeblink
conditioning vis-à-vis the cerebellum (Jacobson et al.,
2005). Although at this time there is no behavioral pheno-
type of FASD, prospective studies of the exposed brains
that seem oddly shaped at birth may well prove a good
strategy for generating one once these children arrive at
an age at which these deficits can be more effectively mea-
sured. In that case, the values for sensitivity and specificity,
after refinement in larger samples, could be referred to the
production of a diagnosis from the FASD range, the deci-
sion of ultimate interest here, rather than to the binary ex-
posure grouping, which is in reality only a probabilistic
cause.
Acknowledgments
This study was conducted as part of the Collaborative
Initiative on FASD, and was funded by Pilot Project grant
number U24 AA014828 from the National Institute on Al-
cohol Abuse and Alcoholism/NIH. Supplemental funding
was received from the University of Washington Alcohol-
ism and Drug Abuse Institute, the University of Washington
Fetal Alcohol and Drug Unit Royalty Fund, and from grant
P200.093/1-VI/2004 from the Austrian Council for Science
and Technology to the Institute for Anthropology, Universi-
ty of Vienna, Austria. Earlier versions of this material were
presented at the June 2004 annual meeting of the Teratolo-
gy Society, Vancouver, Canada; the June 2004 annual meet-
ing of the FAS Study Group, Vancouver, Canada; the 25th
anniversary celebration of the Department of Statistics,
University of Washington, Seattle, September 2004; and
the Konrad Lorenz Institute for Evolution and Cognition
Research, Altenberg, Austria, October 2004.
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