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Neurology Publish Ahead of Print
DOI: 10.1212/WNL.0000000000207114
Sex Hormones and Calcitonin Gene–Related Peptide in Women With Migraine: A
Cross-sectional, Matched Cohort Study
Bianca Raffaelli, MD1, 2; Elisabeth Storch1; Lucas Hendrik Overeem, MSc1; Maria Terhart1; Mira
Pauline Fitzek, MD1; Kristin Sophie Lange, MD1; Uwe Reuter, MD1, 3
Corresponding Author: Bianca Raffaelli, bianca.raffaelli@charite.de
1. Department of Neurology, Charité – Universitätsmedizin Berlin, Berlin, Germany
2. Clinician Scientist Program, Berlin Institute of Health at Charité (BIH), Berlin, Germany
3. Universitätsmedizin Greifswald, Greifswald, Germany
Equal Author Contribution:
This is an open access article distributed under the terms of the Creative Commons Attribution-
NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and
sharing the work provided it is properly cited. The work cannot be changed in any way or used
commercially without permission from the journal.
Published Ahead of Print on February 22, 2023 as 10.1212/WNL.0000000000207114
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
Contributions:
Bianca Raffaelli: Drafting/revision of the manuscript for content, including medical writing for
content; Major role in the acquisition of data; Study concept or design; Analysis or interpretation of
data
Elisabeth Storch: Drafting/revision of the manuscript for content, including medical writing for
content; Major role in the acquisition of data; Analysis or interpretation of data
Lucas Hendrik Overeem: Drafting/revision of the manuscript for content, including medical writing
for content; Analysis or interpretation of data
Maria Terhart: Drafting/revision of the manuscript for content, including medical writing for content;
Major role in the acquisition of data
Mira Pauline Fitzek: Drafting/revision of the manuscript for content, including medical writing for
content
Kristin Sophie Lange: Drafting/revision of the manuscript for content, including medical writing for
content
Uwe Reuter: Drafting/revision of the manuscript for content, including medical writing for content;
Study concept or design; Analysis or interpretation of data
Figure Count: 3
Table Count: 4
Search Terms:
[ 354 ] Gender, [ 100 ] All Headache, [ 101 ] Migraine, CGRP, sex hormones
Acknowledgment:
Study Funding:
The authors report no targeted funding
Disclosures:
The authors report no disclosures relevant to the manuscript.
Preprint DOI:
Received Date:
2022-09-08
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
Accepted Date:
2023-01-11
Handling Editor Statement:
Submitted and externally peer reviewed. The handling editor was Rebecca Burch, MD.
Abstract
Background and Objectives: Sex hormones may modulate CGRP release in the
trigeminovascular system. We studied CGRP concentrations in plasma and tear fluid in
female participants with episodic migraine (EM) and a regular menstrual cycle (RMC),
female participants with EM and combined oral contraception (COC), and female
participants with EM in the postmenopause. For control, we analyzed three corresponding
groups of age-matched female participants without EM.
Methods: Participants with a RMC had two visits: during menstruation on menstrual cycle
day 2 ± 2 and in the periovulatory period on day 13 ± 2. Participants with COC were
examined at day 4 ± 2 of the hormone-free interval (HFI) and between days 7-14 of hormone
intake (HI). Postmenopausal participants were assessed once at a random time point.
Plasma and tear fluid samples were collected at each visit for determination of CGRP levels
with an enzyme-linked immunosorbent assay.
Results: A total of 180 female participants (n=30 per group) completed the study.
Participants with migraine and a RMC showed statistically significantly higher CGRP
concentrations in plasma and tear fluid during menstruation compared to female participants
without migraine [plasma: 5.95 pg/ml (IQR 4.37 – 10.44) vs. 4.61 pg/ml (IQR 2.83 – 6.92),
p=0.020 (Mann-Whitney U test); tear fluid: 1.20 ng/ml (IQR 0.36 – 2.52) vs. 0.4 ng/ml (IQR
0.14 – 1.22), p=0.005 (Mann-Whitney U test)]. In contrast, female participants with COC and
in the postmenopause had similar CGRP levels in the migraine and the control groups. In
migraine participants with a RMC, tear fluid but not plasma CGRP concentrations during
menstruation were statistically significantly higher compared to migraine participants under
COC (p=0.015 vs. HFI and p=0.029 vs. HI, Mann-Whitney U test).
Discussion: Different sex hormone profiles may influence CGRP concentrations in people,
with current or past capacity to menstruate, with migraine. Measurement of CGRP in tear
fluid was feasible and warrants further investigation.
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Introduction
The prevalence of migraine is three times higher in women than in men1. Fluctuations of sex
hormones play a crucial role in the pathophysiology of the disease2. The estrogen-
withdrawal-hypothesis suggests that a drop in estrogen plasma concentrations can trigger
migraine attacks3. In line with this hypothesis, migraine frequency and pain severity are
higher during the perimenstrual phase of the menstrual cycle but also in the perimenopausal
period before hormonal stabilization at an older age2, 4. Migraine prevalence gradually
declines after natural menopause5.
Hormonal contraception leads to the suppression of physiological hormonal fluctuations with
variable effects on migraine6. The most common hormonal contraception in Europe and
North America are combined estrogen-progesterone oral compounds (combined oral
contraceptives, COC)7. While some patients experience an improvement of migraine with
COC, others experience worsening, with migraine attacks occurring most frequently during
the seven-day hormone-free interval (HFI)6.
The pathophysiological mechanisms leading from hormonal changes to the development of
migraine attacks are complex. The neuropeptide Calcitonin Gene-Related Peptide (CGRP)
has a key role in migraine initiation8 and is likely to have a relevant function in the processes
initiated by sex hormones changes. During a migraine attack, CGRP is released from
trigeminal afferents and triggers an inflammatory response9. Preclinical research suggests
that sex hormones fluctuations can lead to activation of the trigeminovascular system and
subsequent release of CGRP, which may contribute to the high prevalence of migraine in
female persons of childbearing age10. However, the clinical evidence in humans is
inconclusive. While older investigations suggest a direct relationship between estrogen and
CGRP concentrations11, 12, newer studies imply a higher CGRP release in low estrogen
phases13, 14.
The accurate measurement of CGRP in peripheral blood is challenging due to its very short
half-life time, degradation, and dilution effects after release15. A recent pilot study detected
increased CGRP concentrations in tear fluid in participants with migraine compared to
control participants without migraine16. This exploratory method is non-invasive and could
provide a more direct measurement of the trigeminal CGRP release due to its spatial
proximity to the trigeminal nerve.
Here, we studied CGRP concentrations in both plasma and tear fluid of female participants
with migraine and female participants without mirgraine under different hormonal conditions.
We aimed to assess the relationship between sex hormones and CGRP levels, and whether
the presence of migraine affects this relationship. It was our hypothesis that a) female
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
persons with migraine display higher CGRP concentrations than female persons without
migraine during the physiological menstrual cycle and b) that the suppression of naturally
occurring sex hormones through COC or after menopause is associated with changes in the
CGRP concentrations.
Methods
Study design and participants
This is a cross-sectional, matched-cohort study at the Headache Center, Department of
Neurology, Charité Universitätsmedizin Berlin, Berlin, Germany.
The study cohort consisted of three groups of female participants with episodic migraine: 1)
With a regular menstrual cycle (M-RMC); 2) Under contraceptive treatment with a COC (M-
COC); 3) During the postmenopause (M-PM). For control, we studied three respective
groups of age-matched control female participants without episodic migraine (C-RMC, C-
COC, and C-PM).
Participants with migraine were recruited from our outpatient headache clinic. For the
recruitment of participants without migraine, we contacted hospital and university staff via
announcements in mailing lists or direct approach.
Inclusion and exclusion criteria
Episodic migraine was defined according to the International Classification of Headache
Disorders 3 (ICHD-3)17. All female participants with migraine should have had at least three
days with migraine in the four weeks prior to screening, as documented in a headache diary.
A RMC was defined as cycle duration of 28 ± 2 days in the three months before screening.
In this group, the diagnosis of menstrually-related migraine17 was required for study
participation. For inclusion in the COC groups, female participants should confirm the regular
use of the same contraceptive drug in a 21/7 regimen (i.e. 21 days of hormone intake [HI]
followed by a 7-day hormone-free interval [HFI]), beginning at least three months prior to
screening. For the postmenopausal groups, the last menstruation should have occurred at
least 5 years before inclusion in the study.
Exclusion criteria were: any other diagnosed primary headache disorder except tension‐type
headache on less than 2 days in the month prior to screening; concurrent migraine
preventive drug treatment; any gynecological or other neurological diseases; ophthalmologic
conditions interfering with lacrimation; any other relevant diseases requiring regular
medication; hormonal treatment with indications other than contraception; pregnancy;
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lactation; post-sterilization. For participants with migraine and a RMC, the diagnosis of pure
menstrual migraine17 led to exclusion from the study.
Study procedures
Before the beginning of experimental procedures, potential participants were screened for
eligibility. Eligible individuals had an initial interview to record their medical history and a
physical examination. In participants with migraine, we reviewed their headache calendars of
the month prior to screening.
The study protocol for female participants with a RMC consisted of two study visits. The first
visit was scheduled at day 2 ± 2 of menstrual cycle (during menstruation), while the second
visit took place at day 13 ± 2 of menstrual cycle (periovulatory period). These time intervals
were selected because estrogen levels are at their lowest during menstruation and at their
highest during ovulation.
Female participants with COC were assessed twice: at day 4 ± 2 of the HFI and between
days 7-14 of HI. Postmenopausal female participants had only one visit at a variable time
point.
All visits in participants with migraine were performed in the interictal period, defined as a
state free of any migraine symptoms and free of acute pain medication for 12 hours before
and after each visit. Participants were instructed to call and reschedule the appointment in
case of migraine or acute medication intake within 12 hours before the scheduled visit. We
also contacted all participants by phone the day after each visit and asked about any
migraine symptoms or medication intake in the 12 hours after study visit. If this was the
case, the visit was repeated at the next possible time point.
Sample preparation and analytical procedures
Each visit took place between 9 a.m. and 5 p.m. in a non-fasting condition. Blood and tear
fluid samples were collected following standardized protocols16, 18.
For CGRP measurement, blood was collected in precooled 4 ml EDTA tubes (BD
Vacutainer®), that were previously prepared with 150 µl aprotinin (3-7 trypsin inhibitor unit
(TIU)/ml) (Sigma Aldrich, Munich, Germany). The tubes were immediately centrifuged for 15
minutes at −6°C and 2000 rpm. Plasma was then transferred in 1.5 ml polypropylene tubes
(Eppendorf, Hamburg, Germany). We collected tear fluid from the lateral canthus of one eye
with a 10µl glass capillary (Brand™, Wertheim, Germany). In participants with migraine, we
selected the eye on the side on which migraine occurred most frequently. If there was no
side preference and in participants without migraine, the right side was chosen by default.
The capillary was removed after reaching the maximal volume of 10µl or after 60 seconds at
the latest. If the eye showed signs of irritation, such as redness or pruritus, the procedure
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was stopped immediately. A lack of tear production after one minute led to exclusion from
the study. The volume of tear fluid collected was determined (range: 1.4 to 10.0 µl) and tear
fluid then transferred in a 1.5 ml tube containing 500 µl of tissue protein extractor solution
(TPER; Pierce Rockford, IL). Both plasma and tear fluid samples were stored at -80°C. We
measured CGRP concentrations in plasma and tear fluid with a commercial sandwich
Enzyme-linked Immunosorbent Assay (ELISA) kit (CUSABIO®, Wuhan, China), following
manufacturer's instructions. The detection range of this kit is 1.56–100 pg/ml, the minimal
detectable dose 0.39 pg/ml. However, the company does not disclose the specific
recognition site of the ELISA antibodies. The kit has high intra-assay and inter-assay
precision (coefficients of variation < 8% and < 10%, respectively). Using this kit, mean
CGRP concentrations in previous cohorts without migraine range from 4.2 pg/ml to 6.6 pg/ml
in plasma16, 19-21 and between 0.7 and 0.8 ng/ml in the tear fluid16, 19.
Additionally, blood was collected in 5 ml serum tubes (BD Vacutainer®) at room temperature
and sent to our partner laboratory (Labor Berlin, Charité Vivantes GmbH) for the analysis of
sex hormones. The following hormones were assessed via electrochemiluminescence
immuno-assay: estradiol, progesterone, testosterone, luteinizing hormone (LH), follicle-
stimulating hormone (FSH).
Endpoints
The primary endpoint of the study was the difference in CGRP concentrations in plasma
(pg/ml) between M-RMC and C-RMC. Secondary endpoints were the differences in CGRP
plasma concentrations between M-COC and C-COC and between M-PM and C-PM.
The differences in tear fluid CGRP concentrations (ng/ml) between the migraine and the
control groups were considered exploratory endpoints.
As further exploratory endpoints, we analyzed correlations between CGRP levels at both
study visits in participants who were measured twice and assessed the differences in CGRP
plasma and tear fluid concentrations among the three migraine and the three control groups.
We also analyzed correlations between the estrogen and progesterone levels and the CGRP
concentrations in tear fluid and plasma.
In addition, the total cohort of participants with migraine was compared with the cohort of
participants without migraine.
Statistical analysis
Sample size calculation was performed using the software G*Power22. Based on a previous
study on interictal CGRP plasma levels in patients with migraine compared to controls
without migraine23, we assumed a large effect size of d = 0.8 for the primary endpoint. A
sample size of 30 participants per group was therefore sufficient to detect an effect of similar
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magnitude with a statistical power of 0.80 at a significance level of α = 0.05 (two‐tailed) using
the Mann-Whitney U test. Similar statistical considerations apply for differences in tear fluid
concentrations16. We therefore aimed at 30 participants per group with complete data sets.
We summarized demographic, anamnestic and laboratory data using descriptive statistics
with median and interquartile ranges (IQR) for numerical variables, and frequencies and
percentages for categorical variables. Given the non-normal data distribution, we compared
outcomes between groups using the Mann-Whitney U test or the Kruskal-Wallis ANOVA, as
appropriate. Correlations were tested using Spearman rank correlations.
Statistical analysis was performed with SPSS Statistics 27 (IBM Corp., Armonk, NY, USA).
No adjustment for multiple comparisons was made for the exploratory outcome measures.
Standard Protocol Approvals, Registrations, and Patient Consents
The study protocol was approved by the Charité Ethical Committee (EA1/004/20). All
participants gave written informed consent following study information.
Data availability
Data not provided in the article because of space limitations may be shared (anonymized) at
the request of any qualified investigator for purposes of replicating procedures and results.
Results
Between August 2020 and May 2022, n=196 persons who self-identified as women
participated in the study. Study protocol was completed by n=180 female participants, n=30
per group. Reasons for drop-out were: no sufficient lacrimation (n=11), occurrence of
migraine in the 12 hours after study visits with no possible rescheduling (n=4) and lost to
follow-up (n=1).
Demographic characteristics were similar between the migraine groups and the respective
control groups. Table 1 shows the demographics across all groups and key migraine
features in the three migraine groups. All female participants with migraine and a RMC
reported migraine attacks within the perimenstrual period during most months17.
Female participants with a regular menstrual cycle
M-RMC and C-RMC presented physiological hormonal levels at the two study visits with low
estrogen concentrations during menstruation and high estrogen concentrations in the
periovulatory period (Table 2). Progesterone levels were low at both time points since both
visits occurred before the luteal progesterone increase (Table 2).
During menstruation, CGRP concentrations in both plasma and tear fluid were statistically
significantly higher in interictal participants with migraine compared to female participants
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without migraine [plasma: 5.95 pg/ml (IQR 4.37 – 10.44) vs. 4.61 pg/ml (IQR 2.83 – 6.92),
p=0.020; tear fluid: 1.20 ng/ml (IQR 0.36 – 2.52) vs. 0.4 ng/ml (IQR 0.14 – 1.22), p=0.005]
(Figure 1).
CGRP levels in the periovulatory period were numerically higher in female participants with
migraine compared to participants without migraine but failed to reach statistical significance
[plasma: 6.28 pg/ml (IQR 3.56 – 9.48) vs. 4.87 pg/ml (IQR 2.95 – 6.41), p=0.089; tear fluid:
0.70 ng/ml (IQR 0.18 – 2.29) vs. 0.63 ng/ml (IQR 0.14 – 1.22), p=0.225].
There was a strong intraindividual correlation between the CGRP concentrations in the
menstrual and the periovulatory visits, both in plasma (rho = 0.809, p<0.001) and tear fluid
(rho = 0.635, p<0.001).
Female participants with combined oral contraception
Both M-COC and C-COC showed suppressed concentrations of naturally occurring sex
hormones. CGRP concentrations in plasma and tear fluid were similar between participants
with migraine and controls without migraine during the HFI and during HI (Table 3). There
was a strong intraindividual correlation between the CGRP concentrations at both visits
(plasma: rho = 0.797, p<0.001; tear fluid: rho = 0.615, p<0.001).
Postmenopausal female participants
Both postmenopausal groups showed physiological hormonal profiles with high
concentrations of LH and FSH and low concentrations of estrogen, progesterone, and
testosterone. There was no statistically significant difference in CGRP concentrations in
plasma and tear fluid between M-PM and C-PM (Table 4).
Comparison of CGRP levels in female participants with migraine in different
hormonal states
Among all participants with migraine, CGRP plasma concentrations were similar among all
groups and visits (p=0.195 among all groups). In the tear fluid, female participants with a
RMC had statistically significantly higher CGRP concentrations during menstruation
compared to female participants under COC (p=0.015 vs. HFI and p=0.029 vs. HI) (Figure
2).
There was no correlation between the absolute estrogen and progesterone concentrations
and the CGRP concentrations in plasma and tear fluid (p>0.17 for all analyses).
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Comparison of CGRP levels in female participants without migraine in different
hormonal states
In plasma, CGRP concentrations of control female participants with a RMC were lower than
those of female participants under COC treatment and postmenopausal female participants
(menstruation vs. HI: p = 0.035; ovulation vs. HI: p = 0.030; menstruation vs.
postmenopause: p = 0.015; ovulation vs. postmenopause: p = 0.013) (Figure 3). No
statistically significant correlation between absolute sex hormone concentrations and CGRP
concentrations could be detected (p>0.17 for all analyses). CGRP levels in the tear fluid
were similar across all groups and all visits of control female participants (p=0.622 among all
groups).
CGRP plasma vs. tear fluid measurements
Across all subjects (n=180) and study visits (n=300), CGRP concentrations were 5.48 pg/ml
(3.98-7.82) in plasma and 0.51 ng/ml (0.16-1.22) in tear fluid. Tear fluid concentrations were
80.5x higher than in plasma (IQR 27.8 – 260.7).
Overall, participants with migraine had statistically significantly higher CGRP levels in tear
fluid compared to participants without migraine [migraine groups: 0.67 ng/ml (IQR 0.17 –
1.59) vs. control groups: 0.41 ng/ml (IQR 0.15 – 0.80), p = 0.013]. Plasma concentrations
were similar with 5.22 pg/ml (IQR 4.03-7.97) in the migraine groups vs. 5.95 pg/ml (IQR 3.73
– 7.79) in the control groups (p = 0.965).
Discussion
CGRP levels in plasma and tear fluid in this large cohort of female participants varied
depending on the presence of migraine and the hormonal status. Female participants with
episodic migraine had higher interictal CGRP concentrations in plasma and the tear fluid
during menstruation than female participants without migraine. This finding did not apply to
female participants with COC and during the postmenopause. In female participants with
migraine, the suppression of the hormonal fluctuations through COC treatment was
associated with lower CGRP tear fluid levels than during physiological menstruation.
Our findings suggest a link between sex hormones and CGRP in migraine pathophysiology
in humans. The influence of sex hormones – in particular estrogen - on intracranial CGRP
release has been studied mainly in vitro or animal research. Estrogen receptors are highly
expressed in CGRP-positive neurons in the trigeminovascular system24 and hormonal
fluctuations can modulate their excitability10, 25. In animal models, deficiency of female sex
hormones increases CGRP expression in various brain regions26-28. Also in the trigeminal
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ganglion, the fall of endogenous estrogen levels in ovariectomized rats led to a significant
increase in CGRP expression, which decreased following estrogen replacement treatment29.
These observations are in line with our results in female patients with migraine: the
physiological estrogen drop in the perimenstrual period was associated with higher CGRP
concentrations than under hormonal contraceptive treatment.
A higher CGRP release during menstruation could help to explain the biological
predisposition for more frequent, severe, and long-lasting migraine attacks in this period30. In
line with this hypothesis, menstrual migraine attacks were more frequent and severe than
non-menstrual attacks even in female persons treated with the CGRP-receptor antibody
erenumab31. Krause et al. (2021) hypothesized that a decline in estrogen levels may lead to
an increased CGRP signaling and generate a pro-migraine state with an increased
susceptibility for migraine attacks25. Of note, this seems to apply only for a decrease in
naturally occurring estrogen concentrations coming from a previously higher level but not for
stable low concentrations during the postmenopause. In addition, the absolute hormone
concentrations do not seem to play a relevant role, but rather the changes in hormonal
levels. Accordingly, all correlation analyses between estrogen or progesterone levels and
CGRP concentrations did not reveal any statistically significant result.
A few older studies showed that sex hormones might affect CGRP concentrations also in
individuals without migraine. Stevenson et al. (1986) detected increased concentrations of
immunoreactive CGRP in plasma during pregnancy, which decreased after delivery11. In a
pivotal study by Valdemarson et al. (1990), CGRP plasma levels were significantly higher in
eleven female participants taking an oral contraception than in twelve female participants
without hormonal treatment12. The study did not provide data on the day of menstrual cycle
or the regimen of hormonal intake12. In accordance with these results, in our study, oral
contraception in female participants without migraine was associated with higher levels of
CGRP in plasma but not in the tear fluid compared to fertile female participants without
contraception. The intake of exogenous hormones seems to induce systemic changes in
CGRP concentrations10, while intracranial CGRP levels as indirectly measured in the tear
fluid seem to be not affected. Indeed, high estrogen states like pregnancy have been
demonstrated to increase CGRP concentrations in other anatomical regions such as the
spinal cord32. Estrogen substitution in rats led to a CGRP increase in the mesenteric
arterioles, dorsal root ganglia33, 34, and in the gastric tract35. Progesterone treatment induced
an increased expression of CGRP receptors in the murine uterus and mesenteric arteries36,
37. The postmenopause is also associated with an increase in systemic CGRP levels38, a
finding which we could reproduce in our cohort of control female participants. The
cardiovascular system has been proposed as the source of the elevated CGRP
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concentrations, as postmenopausal female persons with vasomotor symptoms appear
particularly affected39, 40. Taken together, hormone dependent CGRP changes in plasma of
female persons without migraine seem to originate from sources other than the
trigeminovascular system.
CGRP concentrations in plasma are influenced by a multitude of factors and allow limited
conclusions about the release from the trigeminal nerve system15. It is estimated that only
one fifth of CGRP in peripheral blood derives from trigeminal sources16. While the crucial
role of CGRP in migraine pathophysiology is indisputable, the feasibility of plasma CGRP as
a biomarker of migraine remains a matter of debate15. Previous research reported
controversial results regarding interictal plasma CGRP levels in patients with episodic
migraine: While some studies detected higher CGRP levels in cubital vein blood outside of
acute migraine attacks, others observed no difference to controls without migraine23, 41-43.
Our results provide a differentiated view depending on the hormonal status of the patients.
Female participants with episodic migraine during menstruation had higher interictal plasma
CGRP concentrations than female participants without episodic migraine, while this was not
the case in the other hormonal conditions examined.
Biomaterials closer to the trigeminal CGRP source such as tear fluid may represent a more
direct and suitable approach16. Kamm et al. (2019) reported, in n=30 interictal mix-sexed
patients with episodic migraine, higher CGRP concentrations than in n=48 controls without
episodic migraine16. In the current analysis, we could confirm and expand these findings to a
significantly larger cohort. Similar to this previous study, CGRP levels in the tear fluid were
much higher than in plasma possibly due to lower proteolytic activity in this liquid than in
plasma. In fact, in individuals without ophthalmologic conditions, the levels of peptidases are
generally low in the tear fluid44-46. On the contrary, CGRP in plasma is quickly sheared into
shorter fragment by endopeptidases47, which may in part explain the lower CGRP
concentrations detected with a commercial ELISA. More complex methods such as high-
performance liquid chromatography (HPLC) are able to detect and differentiate between
different peptide fragments47.
CGRP in the tear fluid originates mainly from trigeminal nerve fibers in the cornea and
conjunctiva, while ocular autonomic nerve fibers and the lacrimal and meibomian glands
express only little or no CGRP48, 49. Averaged over the whole cohort, the median CGRP
concentrations in the tear fluid of interictal patients with migraine were higher than in controls
without migraine. This corroborates the hypothesis of an increased activation of the
trigeminovascular system even outside the acute attacks. However, in the analysis by
subgroups, statistical significance was confirmed only in menstruating persons. Future
studies should therefore take the hormonal status of the participants into account when
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
examining CGRP in migraine. Despite these promising findings, CGRP determination in the
tear fluid lacks validation and should be considered as an exploratory procedure. For further
use, a thorough validation study needs to be performed in order to compare performance
characteristics of CGPR levels in the tear fluid with the current standard measurement in
plasma.
This is a comprehensive analysis about sex hormones and CGRP concentrations in female
persons with migraine. The three groups of female participants with migraine were similar
regarding migraine frequency and intensity. The selection of age-matched female
participants without migraine and without other significant diseases or regular medication
represents a key strength of this investigation. The measurement of sex hormone
concentrations at each visit ensured that participants were in the predefined hormonal
phase. Without a continuous hormonal measurement, however, we cannot determine
whether the periovulatory visits took place exactly on the day of ovulation or rather in the few
days before or after. Of note, we excluded female persons with a pure menstrual migraine,
who might possibly have an even stronger influence of hormonal fluctuations on migraine-
inducing mechanisms. Moreover, we included only cisgender women. Therefore, the findings
do not generalize to all women (e.g. transgender women). One further limitation is the
definition of the interictal state, i.e. at least 12 hours free of migraine and acute medication
before and after each visit. This is shorter than in other similar investigations16. We
rationalized that the shortening of this period reduces organizational visit changes and
thereby dropouts. Twelve hours are more than two elimination half-lives of most triptans and
NSAIDs and we did not expect any relevant residual efficacy after this time50. CGRP
measurement requires strict preanalytical sample handling and CGRP concentrations may
vary between studies depending on the exact methodology. In this study, we followed the
protocol by Kamm et al. (2019) with the most sensitive commercial ELISA kit that is
available. Indeed, we found similar concentrations of CGRP in both plasma and tear fluid as
described in this previous study and other studies with the same commercial kit16, 19-21. The
detection of a strong correlation of CGRP levels between study visits in participants that
were assessed twice proves a high interindividual consistency. Importantly, multiple
physiological and pathological processes can influence both CGRP and sex hormone
concentrations. Despite careful selection of subjects and standardized visits, we could not
control for all possible confounding factors. This study is intended as a pilot study. It provides
first evidence of an association between CGRP and different sex hormone profiles in
humans and sets the context for further studies with lager sample sizes and adequate power
to correct for multiple testing and confounders.
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Conclusion
In conclusion, our data suggests hormone dependent changes in CGRP concentrations in
female patients with episodic migraine. The elevated CGRP release from the
trigeminovascular system following hormonal fluctuations could help to explain a higher
susceptibility for migraine in female people who menstruate. The lower CGRP tear fluid
concentrations under hormonal contraception in patients with migraine could be associated
with an altered migraine susceptibility under hormonal therapy and should be further
investigated in a longitudinal design.
Editors’ Note
Neurology recognizes that sex and gender are not interchangeable. Neurology editors aim to
ensure that papers accurately describe and report which of these variables was evaluated in
a study. In this case, the authors included only female participants, and this is the
terminology used throughout the paper. We were unable to find an equivalent term to use in
the title, as style guidelines suggest against using “females” as a noun. Since all the
participants also identified as women, we made an editorial decision to use women in the
title. Neurology strives to affirm persons of all genders and recognizes that the findings of
this article may not pertain to all persons who identify as women.
- Rebecca Burch, MD; Roy H. Hamilton, MD, MS; Holly E. Hinson, MD, MCR
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
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Table 1: Description of the study population.
M-RMC
C-RMC
M-COC
C-COC
M-PM
C-PM
Age (years)
26.50 (24.00-30.00)
26.00 (24.00-31.00)
25.00 (22.75-30.00)
27.00 (22.75-31.00)
57.50 (55.75-60.00)
58.50 (55.75-61.25)
Height (m)
1.69 (1.63-1.74)
1.70 (1.63-1.72)
1.68 (1.65-1.71)
1.69 (1.63-1.74)
1.70 (1.63-1.72)
1.63 (1.60-1.67)
Weight (kg)
63.00 (53.75-73.43)
59.00 (55.00-70.75)
62.00 (56.75-70.25)
59.00 (55.00-70.75)
70.00 (60.75-77.25)
73.50 (62.00-80.50)
Cycle length (days)
28 (27-30)
28 (26-30)
Estradiol dose in COC (mg)
0.03 (0.03-0.03)
0.03 (0.03-0.03)
Progesterone dose in COC (mg)
2.00 (0.15-2.00)
2.00 (0.15-2.00)
Age at menopause (years)
50.00 (48.87-51.00)
50.00 (48.75-52.00)
Age at migraine begin (years)
16.75 (12.37-22.50)
20.00 (17.75-22.13)
20.50 (15.62-31.25)
Aura (n, %)
11, 36.7%
17, 43.3%
9, 30.0%
Monthly migraine days
4.00 (3.87-6.25)
5.80 (4.0-7.0)
5.25 (4.00-9.00)
Pain intensity (0-10 NAS)
7.5 (7.0-8.0)
8.0 (6.0-9.0)
7.0 (6.0-10.0)
Attack duration (hours)
24.00 (12.00-36.00)
27.00 (9.25-48.00)
36.25 (15.75-63.00)
Positive family history (n, %)
22, 73.3%
18, 60.0%
22, 73.3%
Values are median (IQR) or n, %. COC = combined oral contraception. NAS = numeric analogue scale. M = female participants with migraine. C = control female
participants without migraine. RMC = regular menstrual cycle. COC = combined oral contraception. PM = Postmenopause.
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
Table 2: Concentrations of sex hormones in participants with migraine and control participants with a
regular menstrual cycle.
Menstrual
Periovulatory
M-RMC
C-RMC
M-RMC
C-RMC
Day of menstrual cycle
3 (2-4)
2.5 (2-3)
14 (13-15)
14 (12.75-15)
Estradiol (pmol/l)
136.50
(118.75-175.75)
135.00
(99.92-169.25)
576.50
(303.00-961.25)
607.50
(320.75-1019.75)
Progesterone (nmol/l)
0.80 (0.40-1.12)
0.85 (0.50-1.32)
0.85 (0.40-2.42)
0.95 (0.47-2.72)
Testosterone (µg/l)
0.27 (0.18-0.36)
0.24 (0.14-0.34)
0.34 (0.24-0.44)
0.35 (0.21-0.47)
LH (U/l)
5.60 (4.20-6.45)
5.55 (4.00-7.30)
12.35 (7.45-31.95)
15.40 (10.67-30.72)
FSH (U/l)
5.80 (4.72-6.92)
5.80 (4.47-7.22)
6.15 (4.27-9.00)
6.45 (4.57-9.60)
Values are median (IQR). M = female participants with migraine. C = control female participants
without migraine. RMC = regular menstrual cycle. LH = luteinizing hormone. FSH = follicle-stimulating
hormone.
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
Table 3: Concentrations of sex hormones and CGRP in participants with migraine and control
participants with COC treatment.
Hormone-free interval
Hormone intake
M-COC
C-COC
M-COC
C-COC
Day of HFI / HI
3 (2-4.25)
3 (3-4)
10 (8-12)
10 (9.75-12)
Estradiol (pmol/l)
47.65 (20.27-99.70)
21.90 (18.40-58.00)
38.00 (18.40-65.15)
21.30 (18.40-46.03)
Progesterone (nmol/l)
0.30 (0.20-0.50)
0.25 (0.20-0.62)
0.35 (0.20-0.45)
0.40 (0.20-0.70)
Testosterone (µg/l)
0.15 (0.10-0.31)
0.20 (0.13-0.28)
0.14 (0.10-0.23)
0.19 (0.12-0.28)
LH (U/l)
3.20 (0.40-5.32)
1.70 (0.30-4.20)
2.60 (1.20-4.52)
2.15 (0.30-4.90)
FSH (U/l)
3.80 (1.27-7.95)
2.80 (0.30-6.07)
2.55 (1.75-4.12)
1.75 (0.30-4.52)
CGRP in plasma (pg/ml)
4.87 (4.22-6.15)
6.67 (3.76-8.56)
4.92 (3.89-6.24)
6.03 (4.40-9.42)
p = 0.165
p = 0.099
CGRP in tear fluid (ng/ml)
0.46 (0.10-1.01)
0.36 (0.14-0.59)
0.32 (0.09-1.44)
0.40 (0.13-0.82)
p = 0.574
p = 0.690
Values are median (IQR). M = female participants with migraine. C = control female participants
without migraine. COC = combined oral contraception. HFI = hormone-free interval. HI = hormone
intake. LH = luteinizing hormone. FSH = follicle-stimulating hormone.
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
Table 4: Concentrations of sex hormones and CGRP in participants with migraine and control
participants without migraine during the postmenopause.
M-PM
C-PM
Estradiol (pmol/l)
22.80 (18.40-52.30)
28.30 (18.40-47.32)
Progesterone (nmol/l)
0.20 (0.20-0.32)
0.20 (0.20-0.20)
Testosterone (µg/l)
0.11 (0.10-0.19)
0.10 (0.03-0.13)
LH (U/l)
36.10 (28.65-49.77)
37.40 (30.40-44.73)
FSH (U/l)
69.05 (58.70-97.25)
75.70 (61.42-104.25)
CGRP in plasma (pg/ml)
5.24 (3.89-7.14)
6.70 (5.48-8.02)
p = 0.060
CGRP in tear fluid (ng/ml)
0.70 (0.34-1.50)
0.43 (0.21-1.01)
p = 0.280
Values are median (IQR). M = female participants with migraine. C = control female participants
without migraine. PM = postmenopause. LH = luteinizing hormone. FSH = follicle-stimulating hormone.
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
Figure legends
Figure 1: CGRP concentrations in tear fluid (A) and plasma (B) in participants with
migraine and control participants with a regular menstrual cycle (RMC). M = female
participants with migraine. C = control female participants.
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
Figure 2: CGRP tear fluid concentrations in female participants with migraine in
different hormonal states. RMC = regular menstrual cycle. COC = combined oral
contraception. HFI = hormone-free interval. HI = hormone intake. PM = postmenopause.
Copyright © 2023 American Academy of Neurology. Unauthorized reproduction of this article is prohibited
Figure 3: CGRP plasma concentrations in female participants without migraine in
different hormonal states. RMC = regular menstrual cycle. COC = combined oral
contraception. HFI = hormone-free interval. HI = hormone intake. PM = postmenopause.
DOI 10.1212/WNL.0000000000207114
published online February 22, 2023Neurology
Bianca Raffaelli, Elisabeth Storch, Lucas Hendrik Overeem, et al.
Cross-sectional, Matched Cohort Study
Related Peptide in Women With Migraine: A−Sex Hormones and Calcitonin Gene
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