Early Human Development 69 (2002) 91 105

www.elsevier.com/locate/earlhumdev

Methodologic issues in epidemiologic studies

of congenital microcephaly
Alan Leviton a,*, Lewis B. Holmes b,c, Elizabeth N. Allred a,
Juan Vargas d
a

Department of Neurology, Harvard Medical School and Childrens Hospital, Neuroepidemiology Unit,
Box 222, 300 Longwood Avenue, Boston, MA 02115-5724, USA
b
Department of Pediatrics, Harvard Medical School, Harvard, MA, USA
c
Department of Pediatrics, Massachusetts General Hospital and the Brigham and Womens Hospital,
Harvard, MA, USA
d
Department of Obstetrics and Gynecology, University of California, San Francisco, CA, USA
Received 22 October 2001; received in revised form 15 April 2002; accepted 3 July 2002

Abstract
In this methodological paper, we explore a number of issues that pose problems for those who
seek the antecedents of congenital microcephaly. We pay particular attention to three concerns: Who
is a case? How should cases be classified? To whom should cases be compared?
D 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Microcephaly; Newborn; Epidemiology; Growth restriction

1. Introduction
We wanted to identify the second and third trimester antecedents of congenital
microcephaly, and to describe perinatal and neonatal correlates of congenital microcephaly. Unfortunately, a number of methodologic issues became problems that we had to
deal with. We describe here how we handled some of these issues/problems so that others
can avoid some of them, and those who are methodologically inclined can address these
issues and perhaps offer other/better solutions. We also consider other relevant issues.

Corresponding author. Tel.: +1-617-355-6491; fax: +1-617-734-6527.

E-mail address: alan.leviton@tch.harvard.edu (A. Leviton).

0378-3782/02/$ - see front matter D 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 3 7 8 2 ( 0 2 ) 0 0 0 6 5 - 8

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The following comments are intended to help answer three questions and their
components:
(1) Who is a case?
a) Should children with an obvious syndrome be included, especially if the syndrome
tends to be inherited?
b) Should the head circumference cutoff be the lowest 10%, 3%, 2.5%?
c) Should the head circumference standard be internal or external? If external, what are the
most desirable attributes of the standard?
d) Should race-specific standards be used?
(2) How should cases be classified?
a) Should brain imaging be part of the process of classifying cases?
b) What should be done about growth restriction? Should growth restriction dichotomize
the sample? If so, at what point? Should infants with symmetrical growth retardation
(also known as relative microcephaly) be treated as different from infants with
asymmetrical growth retardation (also known as absolute microcephaly)?
c) Should parents head size influence decisions? How should microcephalic infants be
classified it a parents head is small? Should a control infant with a head circumference
on the small size remain a control if one parent has macrocephaly?
(3) To whom should cases be compared?
a) Should referent infants have head circumferences merely above the cutoff for
microcephaly, or should they exceed a higher cutoff (e.g., above the lowest decile)?
b) Should referent infants be matched on gestational age, birthweight or any other
characteristic?
1.1. How should syndromic microcephaly be handled?
One of the most important axioms in epidemiology is to gather incidence cases that are
as homogeneous as possible. In light of current capabilities, congenital microcephaly will
continue to be a rather heterogeneous group of disorders. In an attempt to reduce the
heterogeneity of congenital microcephaly, some have classified congenital microcephaly
into two groups depending on whether or not the microcephaly is accompanied by other
abnormalities [1]. This is reasonable if similar exposures lead to each of the myriad
disturbances that can result in congenital microcephaly. This is not reasonable if the
phenomena that increase the likelihood of trisomy are different from those that increase the
likelihood of translocation.
Infants with a recognizable syndrome have been assumed to have an inherited disorder,
or have suffered the consequences of periconceptional exposures. Recent studies, however, cast doubt on this assumption, suggesting that some microcephalic syndromes (e.g.,
the phenytoin syndrome) reflect later insults [2]. Thus, our decision to exclude infants with
syndromic microcephaly might not have achieved what we had wanted. As it turned out,

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we identified only one microcephalic infant with a recognizable syndrome (Smith

Lemli Opitz syndrome) [3].
Although most children who have a chromosomal disorder have obvious major
physical abnormalities, some have inborn errors of metabolism whose earliest manifestation can be congenital microcephaly [4]. The consequence is that some children who
have no major anomaly are classified as not having a chromosomal disorder, when in fact
they do. Only one infant in our study had metabolic disorder. That infant, whose only
associated anomaly was optic nerve hypoplasia, had a deficiency of 3-phosphoglycerate
dehydrogenase.
We regret that we did not obtain chromosomal analyses of all the infants in our
study who had microcephaly. Until genomic and extensive metabolic analyses are
routine parts of epidemiologic studies of congenital microcephaly, infants whose
congenital microcephaly probably had its origin long before the second trimester will
be inappropriately included with infants whose congenital microcephaly is considered of
unknown origin, and at risk of a disturbance to brain development weeks after the time
of conception.
1.2. Should the head circumference cutoff be the lowest 10%, 3%, 2.5%?
This issue is addressed in detail elsewhere [5]. Microcephaly has been defined as a head
circumference in the lowest decile [6], and as two or more standard deviations below the
mean for (gestational or postnatal) age [7]. A sizable proportion of children with
microcephaly at birth do not have major disability [8]. On the other hand, the more
severe the microcephaly, the greater the risk of later disability [9]. As a consequence, we
chose the more extreme criterion. This decision is supported by Rosenbaums paper [10]
on case definition, which specifically uses microcephaly as an example of the benefits of
power analyses. He compared the power of two case control studies of microcephaly
associated with prenatal diagnostic X-rays. He found that the study had less power when
microcephaly was defined as head circumference in the lowest 2.5% than when microcephaly was defined as head circumference in the lowest 1.5%.
Because every child differs from others in his potential to achieve his own ideal
birthweight, Wilcox et al. [11] suggested that fetal growth restriction be evaluated in
light of each fetus potential to achieve his/her expected weight. They suggested that the
observed birthweight be compared to the expected birthweight based on all the known
factors that influence it, and an individualized birthweight ratio calculated. We raise the
suggestion that the same type of thinking be applied to measurements of head
circumference. Perhaps one day, we will have an individualized head circumference
ratio.
1.3. Should the head circumference standard be internal or external? If external, what are
the most desirable attributes of the standard?
Many of the issues related to the adequacy and choice of external standards (such as
sample size and selection, separate presentations for each sex, exclusions, definitions, and
data quality) are also discussed elsewhere [5]. These issues are not the topic of this paper.

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Our choice of the Oxford UK regional network data set [12] as the external standard
was based on three characteristics. First, the data set includes low gestational age
newborns. Second, the Oxford authors also wisely excluded infants of mothers with
pregnancy-induced hypertension. This decision has little effect among births near term,
when only 2 3% of women have such severe pregnancy-induced hypertension that it is
worthy of the name pre-eclampsia. Near the beginning of the third trimester, however,
infants born to pre-eclamptic women can represent a considerably higher percentage of all
births then [13]. Third, this data set has birthweight distributions that closely approximate
our own. Thus, we are comfortable with the appropriateness of this standard for our own
sample.
We could have used the data of Niklasson et al. [14] from Sweden (Figs. 1 and 2). For
girls born at term, the observed Swedish data are very similar to those of the Oxford UK
group. For preterm girls, however, the formula created by the Swedish group does not
approximate the observed distribution. The formula works better for boys at all gestational
ages, although here, too, the formula gives a lower curve than the observed distribution.
1.4. Should race-specific standards be used?
The rate of premature births (i.e., before 37 weeks) is twice as high in African
Americans (18.4%) as in European Americans (9.1%) [15]. For birth before 28 weeks,
the ethnic disparity is even greater (1.9% vs. 0.5%). African American infants are almost
twice as likely as European Americans infants to be considered SGA based on race-blind
norms [16].
The controversies that accompany discussions of race and gestational age also plague
discussions about race and birth measurement standards [17]. Which contributes more to

Fig. 1. Mean head circumferences for females at different gestational ages (Yudkin standard: solid line; Niklasson
calculation: short dashes; Niklasson standard: long dashes).

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95

Fig. 2. Mean head circumferences for females at different gestational ages (Yudkin standard: solid line; Niklasson
calculation: short dashes; Niklasson standard: long dashes).

the observed differences, race or socioeconomic status? The findings of most studies
indicate that socioeconomic correlates discriminate better than race, and that addition of
these correlates to the list of predictor variables appreciably minimizes the contribution of
race to measurement differences among races [16].
Although birthweight data are readily available for African Americans [17], head
circumference distributions are much less accessible. Using the limited data of Yogman et
al. [18], we found that African Americans and European Americans have very similar
head circumference distributions, except that the curve for European Americans appears
to be shifted to the left by 1 week (at least before the 32nd week of gestation) (Fig. 3). In
essence, the mean head circumference of African American infants at gestational age
X+1 weeks is the same as that of European American infants at age X weeks. This
observation prompted us to apply the Oxford data set to our African American infants,
but shifted 1 week.
We have reservations about this approach mainly because the data are very limited. We
had wanted to extrapolate from observations about birthweight differences between
African Americans and European American infants to head circumference differences.
This may not be appropriate for several reasons.
First, birthweight differences are NOT linear [19]. They are less apparent between the
32nd and 36th weeks than either before or after this gestational age interval.
Second, birthweight can be predicted from a number of maternal characteristics that
tend not to be considered when deciding on whether or not a given infant is growthrestricted [11,20]. For example, every child does not have the same potential to achieve the
mean birthweight for gestational age. Gestational age and race do not predict birthweight
as well as a formula that takes into account such maternal characteristics as age, prepregnancy weight, weight gain, and height, as well as parity and ethnic group. We do not
know if the same or different characteristics predict head circumference.

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Fig. 3. Mean head circumferences for Whites and Blacks at different gestational ages (Yudkin White: solid line;
Yogman White: short dashes; Yogman Black: long dashes).

Thus, we do not feel that in the absence of sufficient amounts of high-quality data we
can justify our decision about head circumference on what is known about birthweight. In
addition, a single uniform adjustment to define expected head circumference differences
also is probably not optimal. Nevertheless, we decided to:
1. accept the total population curve (of Yudkin) for head circumference by gestational age
and label it European Americans, and
2. shift the curve to the right for African Americans by 1 week.
We identify this process as two steps to emphasize that each step deserves evaluation.
We hope that those with a sufficiently large set of high-quality data will evaluate the
appropriateness of our decisions.

2. How should cases be classified?

Congenital microcephaly is not a homogeneous disorder. How one can try to achieve
the most homogeneity possible is the topic of this section.
2.1. Brain imaging
When we planned our study, imaging of the brain was not an integral part of the
evaluation of microcephaly, in part, because most of what was identified did not influence
care. That view still holds in many places. We do feel, however, that future epidemiologic
studies would benefit enormously by including diagnostic imaging as part of the procedure
needed to adequately classify microcephalic cases. Brain imaging at the time of the

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diagnosis of congenital microcephaly can provide information about when the disturbance
leading to the congenital microcephaly had its origin. This would allow grouping of cases
into subgroups classified by likely time of latest exposure to have resulted in the imaged
abnormality. For example, in one series of 33 children identified as microcephalic at birth,
more than 40% had cerebral malformations, most of which reflect a periconception or very
early gestation disturbance [21]. In another series of 30 infants with prenatally diagnosed
microcephaly, more than 80% had the microcephaly as one expression of a more complex
brain disorder, or associated with a chromosomal abnormality, genetic syndrome, or
multiple anomalies [22], again emphasizing the importance of looking for antecedents that
produce their effects near the time of conception.
Brain size determines the head circumference, with shortening of the frontal lobes the
most consistent fetal brain measurement that predicts microcephaly at birth [23]. The
prenatal diagnosis of microcephaly is best made at the end of the second trimester [22,24].
This again supports our hypothesis that much of congenital microcephaly has its origins
long before the end of the second trimester.
Imaging the brains of microcephalic cases but not the brains of referent newborns is
unlikely to result in ascertainment bias, mainly because the abnormalities identified in cases
occur so rarely in those who are not microcephalic. Investigators also need to consider the
ethical issue of asking a family to consent to a procedure that is unlikely to provide any
benefit for the newborn, and the resistance of most parents to consent to such a procedure.
2.2. What to do with growth-restricted infants?
Microcephaly is over-represented among newborns considered growth-restricted
[25,26]. If the growth restriction involves the head as much as body, then the growth
restriction is considered symmetrical. Others prefer the term relative microcephaly to
describe the small head on a small child and absolute microcephaly to describe the small
head on a normal-size child [27]. Some claim that relative microcephaly is associated
with a better intellectual prognosis than absolute microcephaly [27].
Some of this concern about small body is based on the axiom that the brain should be
spared when inadequate nutrition contributes to restriction of body weight gain [28]. It is
not clear if the brain should also be spared when the brain is exposed to a neurotoxin.
The over-representation of microcephaly among growth-restricted infants leads to the
concept of two kinds of microcephaly, one associated with growth restriction, and one not
associated with growth restriction. This prompted us to explore the antecedents of
microcephaly separately among the growth-restricted, and among those whose birthweight
is considered appropriate for gestational age. The problem with this approach is best
appreciated by an examination of Figs. 4 6. Before examining these figures, however,
please read the following paragraph.
To avoid problems with adjusting for gestational age, we assigned each infant a Z score
for birthweight and head circumference based on his/her position along the sex-specific
distributions for gestational age. The Z score is the number of standard deviations above or
below the mean. A Z score of 2 indicates that the infants measurement is 2 standard
deviations below the mean, which is the cutoff we chose for both growth restriction (i.e.,
birthweight) and microcephaly.

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Fig. 4. Scatter plot of head circumference Z scores and birth weight Z scores. Each small circle represents a baby
in this sample. The location of each circle identifies the babys head circumference Z score and birth weight Z
score. The horizontal and vertical solid lines, which identify Z scores of 2, divide this scatter plot into four
quadrants that define babies as they are classified in Figs. 5 and 6. Babies to the right of the vertical solid line are
not small for gestational age (S ), whereas those to the left of this line are small for gestational age (S+).
Similarly, those above the horizontal solid line are not microcephalic (M ) and those below the horizontal solid
line are microcephalic (M+). Babies identified as M S are those in the upper right quadrant because they are
neither microcephalic nor small for gestational age. Continuing in a clockwise fashion are the other three
quadrants representing babies identified as M+S , M+S+ and M S+.

Fig. 5. Cumulative percentage distributions of birth weight Z scores for each of the four groups identified in Fig. 4.

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Fig. 6. Cumulative percentage distributions of head circumference Z scores for each of the four groups identified
in Fig. 4.

In our data set of both normal weight and growth-restricted infants we, too, found that
the lower the body weight Z score, the lower the head circumference Z score (Fig. 4). In
essence, the lower the infants birthweight compared to peers (i.e., others of the same sex
and gestational age), the smaller the head circumference. Among growth-restricted infants
(i.e., those to the left of the solid vertical line), most infants had microcephaly (i.e., those
below the solid horizontal line). This is clearly not the case among infants with higher
birthweight Z scores.
Cumulative percent distributions facilitate appreciation of differences and similarities
[29]. We compared the cumulative birthweight distributions of infants in each of the four
quadrants in Fig. 4 created by the solid lines (Fig. 5). Among microcephalic, growthrestricted infants (M+S+), those in the lowest birthweight quartile have appreciably lower
birthweights than normocephalic, growth-restricted infants (M S+). It is as if this group
of the most growth-restricted infants is microcephalic by definition.
Among infants whose birthweight was appropriate for gestational age (S ), those who
were microcephalic at birth (M+) had considerably lower birthweights than their
normocephalic peers (M ). This raises the possibility that even among appropriate for
gestational age infants, the antecedents contributing to microcephaly also influence body
weight.
The cumulative sum distribution of infants classified by their head circumference Z
scores shows minimal difference between those in the microcephalic, growth-restricted
group (M+S+) and those in the microcephalic, appropriate for gestational age group
(M+S ). Yes, the head circumference Z score distribution for growth-restricted
infants is to the left of the appropriate for gestational age infants, but not by much.
On the other hand, among normocephalic infants, the cumulative sum distribution of
head circumference Z scores for growth-restricted infants is considerably to the left of
the distribution for appropriate for gestational age infants. A reasonable inference here

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is that the antecedents limiting weight gain also limit head circumference growth. If
head circumference is a surrogate for brain growth (and this is acknowledged to be
controversial), then the inference becomes the antecedents limiting body growth also
limit brain growth.
If, when seeking to identify the antecedents of microcephaly, we stratify our sample by
birthweight into growth-restricted and appropriate for gestational age, we are faced with
several problems. One is a non-specific problem that follows from stratification in general.
We believe strongly in stratification strategies when scrutinizing data. Nevertheless,
stratification into growth-restricted and appropriate for gestational age subgroups results
in a loss of power that follows from splitting a sample into parts.
The other main problem results when the stratification is by a variable that might
itself be a marker of causal chain events. Consider the likely possibility that reduced
birthweight is a marker of disturbances to fetal well-being. Selecting the referent group
to be like the cases in this characteristic results in overmatching [30]. This happens
when the referent group is too similar to the cases. In essence, by group-matching on
birthweight Z score, we limit our ability to identify antecedents of microcephaly that
are associated with growth restriction. For example, early second trimester cytomegalovirus infection leads to fetal growth restriction and microcephaly [31]. Matching on
birthweight would limit our ability to perceive a relationship between microcephaly
and antecedents that also lead to growth restriction, such as cytomegalovirus infection.
2.3. Familial tendencies
Microcephaly vera, the disorder defined as familial microcephaly, is viewed as a benign
disorder without increased risk of cognitive limitations [32]. However, this heterogeneous
group of disorders is not invariably benign [33]. Segregation analyses suggest that
approximately half of all microcephaly cases seen in genetic counseling clinics are due
to highly penetrant recessive mutant alleles [34]. Recently, isolated familial microcephaly
has been mapped to five loci [35 39].
Familial microcephaly raises several issues that have tended to be ignored in defining
congenital microcephaly:
(a) Should parents head circumferences be measured on all infants, and these
measurements entered into equations of phenomena that predict infants head circumference at birth?
We were able to obtain head circumference measurements on the newborns mother
for 84% of our AGA controls. In this sample of AGA controls, we created linear
regression equations of infants head circumference as a function of the mothers head
circumference alone (Fig. 7A). With every centimeter of decrease in the mothers head
circumference, the babys head circumference Z score diminished by 0.7 (standard
deviation units). Although small, this differs significantly from zero in this sample.
Thus, we infer that the mothers head circumference makes a small, but discernable
contribution to population variances in newborns head circumference. Although we had
fewer measurements of fathers head circumference, the relationship between fathers
and infants head circumferences approximate those seen between mother and infant
(compare Fig. 7B to Fig. 7A).

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Fig. 7. (A) Babys head circumference (ordinate) plotted against mothers head circumference (abscissa). The line
gently sloping upward represents the linear regression of babys head circumference on mothers head
circumference. (B) Babys head circumference (ordinate) plotted against fathers head circumference (abscissa).
The line gently sloping upward represents the linear regression of babys head circumference on fathers head
circumference.

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(b) How should infants head circumference be evaluated when parents head circumferences have not been measured? Will use of dummy variables for missing data be
adequate?
As described above, we are not opposed to an individualized head circumference ratio.
Based on our own data, however, we are not sure that information about the parents head
circumferences will help the clinician calculate the infants own individualized head
circumference ratio.
(c) Should infants with one microcephalic parent (defined as head circumference in the
lowest 3% or 10%) be considered in our case groups of presumably acquired microcephaly?
The familial characteristic of small head circumference is usually labeled microcephaly vera. We prefer the descriptive term, familial microcephaly. Children with
familial microcephaly tend not to be at increased risk of cognitive limitations [32]. Thus,
this familial characteristic is not viewed as a disorder. More importantly, it is unlikely to
represent exogeneous exposures during pregnancy. Including infants with familial microcephaly in a case group of infants with presumably acquired congenital microcephaly will
result in misclassification of some infants as cases. We, therefore, consider it prudent to
exclude such infants from the case groups. Doing so will sometimes pose problems.
Familial microcephaly, even when occurring in siblings, can be associated with abnormalities of anatomy (extremely thin, smooth cortex) and function [40]. It seems reasonable
to conclude that not all familial microcephaly is benign, and that familial microcephaly is
probably a heterogeneous entity. The potential for misclassification is high.
Would the infant with a head circumference just above the microcephalic criterion (i.e.,
in the 3 10% range) have been microcephalic if he had a macrocephalic parent? Should
he be excluded from the control group because he would have had a head circumference
below the third centile if both his parents were normocephalic? What inferential price
would we pay if we excluded infants from both case and control groups if they had a
parent with either micro- or macrocephaly? Should we suggest that any infant who has a
parent with a head circumference in the lowest or highest X% be excluded from both the
case and the control group?

3. Characteristics of referent newborns

The previous section raises the issue of the characteristics of the referent group. The
basic question here is, Where should the arbitrary cutoffs be for controlness when
caseness is also defined by an arbitrary dichotomization of a continuous measure? Should
the referent infants be as normal as possible? Should their head circumferences be within
the 10th 90th centile bounds? If all the microcephalic cases have head circumferences in
the lowest centile, can the referent infants have a head circumference above the third
centile?
3.1. Should referent infants be matched to cases?
Some of the issues related to the question of matching have already been discussed in
the section above dealing with growth restriction. Here we expand on the basic point that

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matching or stratifying on a characteristic that is associated with antecedents of congenital

microcephaly impedes our ability to identify correlates of the matching variable.
With biomarker epidemiology gaining acceptance, the time will come when epidemiologic studies of microcephaly will be based on routine measurements of protein concentrations or gene expression of growth factors. Such growth factors as insulin-like growth
factor I (IGF-1) [41 43], nerve growth factor [44], vasoactive intestinal peptide [45], and
glucose transporter protein [46] all restrict fetal body and head growth. Matching on
birthweight, in essence matching on fetal growth restriction, will diminish the opportunity
to identify such growth promoters as antecedents of congenital microcephaly. Yes, lacking
the gene for one of these growth factors has profound importance to the individual and his
family. On the other hand, minor to moderate deficiencies of these proteins might have
considerable importance on a population basis. For example, even among normal term
singletons, the lower the umbilical cord blood concentration of insulin-like growth factor I,
the smaller the head circumference [47].
Another example of overmatching also comes from our own growth-restricted strata.
The infants in the referent normocephalic group have smaller heads than the normocephalic infants who serve as referents for microcephalic, appropriate for gestational age
infants (Fig. 7). This is not a problem if we have good reason to believe that small
infants should have small heads. If, however, we question this assumption, then selecting
growth-restricted normocephalic infants to be the referent group for microcephalic
growth-restricted infants will limit our ability to perceive some phenomena that increase
the risk of microcephaly.
4. Conclusions
We have asked more questions than we have answered. This reflects our inability to
clarify some ambiguities. Perhaps our limitations are not unique. Perhaps the inability of
others to confidently answer some of the questions we raise accounts for the paucity of
epidemiologic studies of congenital microcephaly. Until consensus has been achieved on
how to handle these issues, we ask that students of microcephaly epidemiology clarify
their decisions about sample selection, including all inclusions and exclusions, as well as
about classifications. We also hope that increases in our understanding of the biology of
head growth, as well as improvements in genetics and brain imaging, will allow better
studies to be conducted.
Acknowledgements
This study was supported by the National Institutes of Health (NS26093).
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