Supplementary Information
Analysis of Functional Connectivity
The functional connectivity analysis was seeded from regions having different cortical thickness.
While this could in general bias the results, we believe in our study this was not the case since
the same seed mask was used in all of the subjects and the magnitude of the observed
differences in the cortical thickness is 10th of mm (plots in figure 3b, ~0.2mm for precuneus, and
~0.4mm for the insula in female vs. male migraineurs), which is well below the resolution of the
fMRI images (3.5 x 3.5 x 3.5 mm3).
Other Regions showing sex differences
Hypothalamus
Sex-related differences in hypothalamic function (deactivation: male > female) were also
defined. The hypothalamus has extensive and multiple functions in the brain (Sprenger et al. ,
2004, Overeem et al. , 2002, Moore, 1997, May et al. , 1998), and it appears involved in migraine
pathophysiology (Denuelle et al. , 2007) even though its exact role in migraine remains unclear
(Alstadhaug, 2009). Given the role of the hypothalamus in hormonal regulation, it is perhaps
not surprising that we also found differences between men and women. This difference is also
striking given that we did not assess the menstrual cycle stage for the female. The differences
observed may relate to alterations within each migraine group with women showing a decrease
and men showing an increase. Increased activation in men is consistent with other functional
imaging findings of headache such as cluster headache (May et al. , 1998). As the trigeminal
nucleus (SpV) also showed a stronger response to noxious stimulation in women, we believe
that the activations observed in the study were from nociceptive information conveyed by the
trigeminohypothalamic pathway to the hypothalamus (Malick and Burstein, 1998).
Hypothalamic responses may also be a result of altered stress/autonomic responses (Goldstein
et al. , 2010, Nunn et al. , 2011) or hypothalamic priming resulting from differences in cyclic
hormonal processes (Goldstein et al. , 2005).
Basal Ganglia
Alteration in morphology and function of the basal ganglia is present in migraineurs (Maleki et
al. , 2010a). The basal ganglia play an important role in multiple functions (Herrero et al. , 2002)
including pain processing (reviewed in (Moulton et al. , 2011)). In the present study, we
observed deactivation of nucleus accumbens (NAc) for both male and female migraineurs in
response to painful stimulation that was more significant in men. We also found stronger
activation responses in bilateral caudate (F>M) and contra-lateral putamen (M>F). In our
previous studies we had observed altered functional connectivity in the caudate of high vs. low
frequency migraineurs (7 female: 3 male in each cohort) and we also had observed decreased
volumes in adult migraineurs with high frequency vs. low frequency migraines (Maleki et al. ,
2010a). While alterations in basal ganglia circuits are common in pain imaging studies (Borsook
et al. , 2010) and reports of alterations in iron accumulation in migraineurs in the putamen and
globus pallidus (Kruit et al. , 2009), the reason for the sex differences are unclear. Putaminal
activation may correlate with an anti-algesic or analgesic process since it is also activated in
phMRI analgesic studies (Upadhyay et al. , 2011) while prior pain or migraine imaging studies
have not reported specific sex differences in the basal ganglia. Pathways that originate in the
trigeminovascular systems may send collaterals to the basal ganglia (Noseda et al. , 2011), but it
is not known how these may impact on sex differences except that differences in
neurotransmitter systems may be present in men and women as described in other pathological
conditions affecting the brain (Tayoshi et al. , 2009). Also noted were differences in the ventral
striatum (nucleus accumbens) where greater deactivation was observed for men vs. women. We
have observed deactivation in the region in response to heat in healthy subjects and have
interpreted this as a measure of hedonic responses to aversive stimuli (Becerra et al. , 2001,
Becerra and Borsook, 2008). In chronic pain patients, the activity in the region in response to
noxious heat may encode predicted value of pain and anticipates its analgesic potential on
chronic pain (Baliki et al. , 2010).
Posterior Cingulate
Significant differences in BOLD responses to pain were observed in the posterior cingulate with
activations for women greater than men (Table 3). The posterior cingulate receives afferent
inputs from the anterior thalamic nuclei and from extensive cortical areas in the frontal,
parietal, and temporal lobes (Vogt et al. , 1979). It has been suggested that the region is
involved in components of a self-reflection/self-regulation (Johnson et al. , 2006). Alterations in
posterior cingulate volume in healthy subjects (greater in women) (Mann et al. , 2011) and in
migraine (smaller in healthy) have previously been reported (Kim et al. , 2008). In the latter
study (Migraine: 20 (3 male/17 female) vs. Healthy Control 33 (4 male/29 female)), this was
reported to correlate with decreased metabolic measures in the same area (Kim et al. , 2010).
The posterior cingulate is also activated by light (Boulloche et al. , 2010) and by olfactory stimuli
in interictal migraineurs (Demarquay et al. , 2008) suggesting that it may be part of brain regions
sensitized in the migraine condition. Activation as demonstrated by PET imaging during migraine
has also supported posterior cingulate activation (Afridi et al. , 2005). Again, the present finding
that female migraineurs had a more reactive posterior cingulate than male migraineurs may
indicate more severe disease consequences in women.
In contrast to areas involved in emotional processing of pain, we also observed changes in the
primary somatosensory cortex (SI). Previous fMRI studies on sex differences in healthy subjects
indicate that men have greater pain activation of the somatosensory cortex (Derbyshire et al. ,
2002, Moulton et al. , 2006, Paulson et al. , 1998, Straube et al. , 2009, Kong et al. , 2010). Our
results also show significant difference in the pain activation (women < men) of the contralateral
somatosensory cortex while no differences were observed in the ipsilateral SI. This observation
was seen even though no differences in pain intensity were measured between the two
migraine groups. We interpret this difference in terms of other systems that modulate or
dampen pain intensity including those abnormalities discussed above.
Study limitations
Measure of Menstrual Period
Although menstrual cycle stage was not measured in these experiments, the addition of
a monthly menstrual related pain may also further sensitize or aggravate pain circuits in women
independent of whether their migraine is paired with the onset of their menstrual period.
Future studies should utilize repeated measures across the menstrual cycle and/or measure
gonadal hormone levels, as well as detailed exploration of sex related pain expectations
(Vierhaus et al. , 2010).
Head Size
The issue of head size and sex is complex when utilizing MRI measures of gray matter volume
may be a confound with between-subject comparisons (Barnes et al. , 2010). As it is known that
men generally have larger volumes and thinner cortex than women, we adjusted for this by
normalizing volumes. Specifically, structural neuroimaging of healthy humans also report gray
matter differences between men and women areas of the cortical mantle are thicker in women
than in men (Cahill, 2006), particularly in the posterior medial wall (Luders et al. , 2006).
Women have larger volumes, relative to cerebrum size, in frontal and medial paralimbic
cortices, where as men have larger volumes, relative to cerebrum size, in frontomedial cortex,
the amygdala and hypothalamus (Goldstein et al. , 2001). In this study we compared matched
healthy men and women to account for such disparities. In doing so, none of the changes
reported in the migraine groups were observed.
Number of Subjects
The number of subjects studied is relatively small. There was a three-way match for the study:
migraine men and women (age, migraine frequency, medications) and for migraine sex and
healthy controls (age and sex). However all analysis, including structural MRI or fMRI datasets,
were performed using rigorous statistical tests. Specifically, for fMRI we implemented a mixedeffects group analysis to incorporate individual variability into the second level (cohort-level)
statistics. We performed inference utilizing robust adaptive techniques, such as, Gaussian
mixture modeling to determine statistical significance minimizing false positive as well as false
negatives. Morphometric statistical inference were performed based on standard techniques
(general linear model) and the vertex-based cortical thickness comparisons were corrected for
multiple comparisons using a Monte-Carlo based simulation for multiple comparisons.
Other Structural Abnormalities
Structural changes associated with migraine are not limited to the gray matter and multiple
studies have reported either the presence of white matter abnormalities of the brain of migraine
patients in the form of focal hyperintense lesions using magnetic resonance imaging or changes
diffusion properties (ex. reduced fractional anisotropy (FA)) of specific white matter fibers (ex.
optic radiation (Rocca et al. , 2008), trigeminothalamic white matter tract (DaSilva et al. , 2007))
in migraineurs without aura have lower fractional anisotropy in the ventrolateral periaqueductal
grey matter. While We did not explore white matter differences in these cohorts, there hasn’t
been any report on gender specific differences in white matter abnormalities and may be that's
something that should be explored in the future studies.
Medication Use
Finally, as with many studies that compare healthy controls with a disease state, the issue of
medication use remains a confound. In this study, migraine some patient were on either
triptans or non-steroidal anti-inflammatory drugs (NSAIDS). In comparing male and female
migraineurs, we tried to control for this confound by matching subjects in terms of the type and
the history of the medications that they had used. However for the comparisons between
migraineurs and healthy control subjects, we cannot rule out direct triptan-mediated effects.
Supplementary Figures
Supplementary Figure 1: Direct within sex comparisons. Significant clusters from vertex-wise
with gender cortical thickness comparisons for disease-only effect in female healthy vs. female
migraineur subjects (left column) and in male healthy vs. male migraineur subjects (right
column). The blue-light blue colors represent areas with cortex thicker in female migraineurs vs.
female healthy control subjects. There were no significant differences in cortical thickness
between male migrianeurs vs. male healthy control subjects.
Supplementary Figure 2: Functional Connectivity in Male and Female Subjects. The blue-light
blue colors represent areas with negative correlation and red-yellow colors represent positive
correlations between insula and precuneus with the rest of the brain.