Increased serum prolactin level may indicate more migraine attack frequency

Abstract Objectives Migraine is a common, multifactorial disorder. The exact pathomechanism of migraine remains unclear. Studies have revealed changes in serum prolactin (PRL) levels in relation to migraine, although the results have been inconsistent. The present case‐control study assessed the serum level of prolactin in migraine patients. Materials and methods In this case‐control study, participants were divided into chronic migraine (CM; n = 39), episodic migraine in ictal (during an attack), and interictal (between attacks) phases (n = 63, n = 37, respectively) along with 30 age‐ and sex‐matched headache‐free controls. After obtaining demographic, anthropometric data, and headache characteristics, blood samples were gathered and analyzed to evaluate the serum levels of prolactin (ng/mL). Results A significant difference was observed between the control, CM, and ictal EM, and interictal EM groups. The mean ± SD serum prolactin levels of the chronic migraineurs (1.82 ± 0.94) and those with ictal EM (1.93 ± 1.70) were comparable and were significantly higher than for interictal EM patients (0.82 ± 0.46) and the headache‐free control subjects (0.49 ± 0.15; p < .001). Although the mean serum concentration of prolactin for the interictal EM group tended to be higher than for control individuals, this difference was not statistically significant. The Spearman's correlation test also showed significant correlations between the serum prolactin levels and the number of headaches days among migraineurs. Conclusion The findings suggest that there might be an association between increased prolactin concentrations and migraine headache induction and progression. Further detailed and well‐designed studies are needed to confirm the importance of serum prolactin levels in the pathogenesis of migraine headaches.

Extreme sensitivity to light, sound and smell along with vomiting and nausea are typical migraine symptoms (Edvinsson et al., 2018).
Migraine is the most prevalent medical disorder and the second leading cause of disability globally (Ashina et al., 2021).
Migraine patients can be classified as either chronic migraine (CM) or episodic migraine (EM). An EM is characterized as fewer than 15 migraine headaches per month. CM is characterized as more than 15 headache days per month over the course of 3 months, of which at least eight days have migraine characteristics or show response to triptans (Headache Classification Committee of the International Headache Society [IHS], 2018). On average, migraine occurs in 15% of the population in 1 year (Ashina et al., 2021) with a marked sex difference.
Migraine is two to three times more prevalent in females than in males (Tire et al., 2019;Woldeamanuel & Cowan, 2017).
Several factors cause sex differences in migraine, one of which is sex hormones such as estrogen, progesterone, and prolactin. There is increasing evidence that a higher level of prolactin in women can promote pain. In addition, it may be because the expression of a prolactin receptor in female nociceptors and their responses to external stimuli are different (Gazerani, 2021). Although the exact pathophysiology of migraine headache is unknown, it has been shown that it appears to occur in relation to a mixture of environmental and genetic factors (Goadsby et al., 2017). It is likely that migraine attacks are caused by both peripheral and central mechanisms. It remains unclear whether migraine attacks begin in the peripheral or central nervous system (Do et al., 2023). Genetic changes in ion channels, the trigeminal release of CGRP from C-fibers, and cranial vasodilatation in the central nervous system (CNS) may have roles in initiating migraine attacks (Do et al., 2023;Edvinsson et al., 2012). Several events in the trigeminal ganglion can give rise to migraine attacks, including neuronal hyperexcitability in the cortical region, cortical spreading depression (CSD) leading to the activation and sensitization of trigeminal nociceptors, but is still controversial, and cranial vasodilatation and the release of inflammatory molecules in the meninges (Artero-Morales & González-Rodríguez, 2018; Charles, 2018;Zhang et al., 2010).
Migraine attack can be triggered by several factors that include stress, changes in sleep patterns, specific sensory stimuli (light, sound, and smell), alcohol, foods, and hormonal fluctuations (Gazerani & Cairns, 2018). Hormones that act on the pituitary-hypothalamic axis are thought to be important in the pathophysiology of migraine. Of this axis, prolactin, a polypeptide composed of 199 amino acids, is secreted from the lactotrophic cells of the anterior pituitary gland (Saleem et al., 2018). Prolactin secretion is controlled by dopamine, which is a inhibitory regulator (Saleem et al., 2018) and is positively regulated by stress, exercise, BMI, circadian rhythms, serotonin and proinflammatory cytokines such as TNF-α and IL-6 (Corona et al., 2011;Fojtíková et al., 2010;Roelfsema et al., 2012).
Studies have shown that there is an association between increased prolactin levels and blood pressure, cardiovascular disease, diabetes type 2 (Corona et al., 2011), rheumatoid arthritis (Fojtíková et al., 2010), inflammatory diseases, pain (Patil et al., 2013), and neurological diseases such as headache (Pineyro et al., 2017) and multiple sclerosis (Wei et al., 2017). It is interesting to note that the level of prolactin has been associated with some CNS manifestations and disease activity.
Reports about the level of prolactin in migraineurs are conflicted.
Some studies have reported that serum prolactin levels seem to be higher in migraineurs than in headache-free subjects Guldiken et al., 2011;Noori-Zadeh et al., 2020). Other studies have reported contrary results (Masoud & Fakharian, 2005;Murialdo et al., 1986). Furthermore, most studies have been based on small sample sizes and did not examine different categories of migraine (Alia et al., 2013;Bosco et al., 2008;Masoud & Fakharian, 2005). The evidence suggests that it is necessary to determine the exact role of prolactin in the pathogenesis of migraine with the use of well-designed studies having sufficient sample sizes. To this end, we designed a study to assess serum prolactin levels in subjects with EM and CM and compared these levels with an age-and sex-matched headache-free control group to investigate the relationship between prolactin and migraine.

Study population
This case-control study examined chronic migraine (CM; n = 39), episodic migraine in ictal (during an attack) and interictal (between attacks) phases (n = 63, n = 37, respectively) and 30 headache-free ageand sex-matched control subjects. Recruitment was from September 2017 to June 2020. The migraine groups were recruited from the Sina University Hospital Headache Clinic (a tertiary headache clinic) and the control group was selected from age-and sex-matched headache-free candidates from the general population (hospital staff and patient companions). Individuals in both groups were chosen from respondents to an advertisement (the posters were mostly displayed in headache clinics and their immediate surroundings, but they were also displayed throughout the hospital) for voluntary participation in a case-control study on the serum level of prolactin in migraine.
A neurologist who is an expert headache specialist diagnosed the

Headache diaries and visual analog scale
At the beginning of the study, the required information about demographic and anthropometric characteristics, medications, and medical history were collected from the participants. After that, blood samples were collected from the control subjects. The migraine subjects were diagnosed according to ICHD3 criteria by the headache specialist (Prof. M.T.), who was the senior investigator in this research.
The migraine subjects were then instructed to fill out a headache diary during the month ahead (Razeghi Jahromi et al., 2018). A headache diary records the number of headache days, the duration, and severity of each headache attack, and the number of times abortive drugs were used. Participants were contacted by telephone weekly during the month. On their second visit after 1 month, at the time the headache diaries were collected, blood samples were taken from the CM and EM subjects in the ictal phase (n = 63) and EM subjects in the interictal phase (n = 37) (72 h after most recent attack). In the case of ictal phase, patients were instructed not to take their usual painkillers or specific migraine medication until blood samples were taken.
The mean severity of the headache was measured by using the visual analog scale (VAS), which rates pain intensity from 0 (almost no pain) to 10 (worst possible pain experienced by a person).

Serum prolactin assessment
The blood samples (10 mL

Basic characteristics of migraine subjects and control group
The basic characteristics of the subjects are summarized in Table 1.
No significant differences were observed in the distributions for age, sex between groups. From 30 subjects in the control group with mean ± standard deviation (SD) age of 41 ± 8, 73.3% were women.
78.4% of the interictal EM, 87.3% of the ictal EM subjects, and 74.4% of the chronic migraine patients were female. The mean age of the chronic migraine, ictal EM, and interictal EM were 39 ± 8, 38 ± 8, and 37 ± 12, respectively.
Regarding BMI, significant differences were found between groups (p = .038). Table 2 shows the headache characteristics of the three migraine groups. The severity of headache according to VAS criteria was significantly higher in CM than in EM subjects. There were no significant differences in the duration of attacks between groups.

Serum prolactin levels in studied groups
The serum levels of prolactin in the four groups are shown in Table 3 and Figure 1 showed significant correlations between serum prolactin levels and the number of headache days for migraineurs ( Figure 2).

DISCUSSION
In the current study, we compared the level of prolactin in migraineurs with age-and sex-matched headache-free subjects and investigated its association with the number of headache days. It was shown that prolactin levels in subjects with ictal EM did not differ significantly from those with CM. A major finding was that both CM and ictal EM subjects had significantly higher prolactin levels than the interictal EM and control subjects. It was also found that interictal EM subjects out of the acute phase had higher levels of prolactin than the headache-free subjects, but the difference between groups was not significant.
Significant correlations were found between the serum prolactin levels and the number of headache days among migraineurs (p = .002).
Although studies evaluating prolactin levels in subjects with migraines have shown different results, taken as a whole, our results on the levels of prolactin in migraineurs compared to controls appear to be corroborated by previous evidence in this field (Cavestro et al., 2006;Li et al., 2018;Nattero et al., 1986;Taha & Mohammed, 2019;Vega et al., 2017). Li et al. (2018) assessed serum different hormones related to female gonadal function including prolactin of migraineurs and analyzed the relationship between these hormone levels and migraine characteristics. The serum levels of these hormones were compared between 119 migraineurs, 42 subjects with tension-type headaches, and 30 headache-free control subjects. They concluded that the levels of prolactin were significantly higher in migraineurs than the control group.
The findings of the present study showed a lack of significant differences between the interictal EM group and control group. high-sensitivity C-reactive protein (hs-CRP) in migraineurs between attacks. In accordance with our findings, they found no a significant difference between the prolactin levels of migraine subjects in their interictal phase and the controls.
The outcomes of some studies which reported that no significant difference was found between prolactin levels in migraineurs compared to the control group were not consistent with those of the present study (Guldiken et al., 2011;Seddighi & Dehghani, 2002). Seddighi and Dehghani (2002) gauged the relationship between acute migraine attacks and changes in prolactin in 20 subjects with migraine and 20 subjects with nonmigraine headache. As opposed to our study, they concluded that serum prolactin was not elevated in the acute phase of migraine. In contrast to the present study, Masoud and Fakharian (2005) who measured serum prolactin levels during acute attacks in 37 subjects with migraine and 37 with nonmigraine headaches reported that prolactin levels decreased during the acute migraine headaches. One of the reasons for the difference in conclusions in these studies compared to the present study could be their small sample size. Another reason could be the lack of separate examinations of prolactin levels in subjects with EM and CM.
It is worth mentioning that most previous studies only evaluated the difference of prolactin level among migraineurs and a headachefree control group (Cavestro et al., 2006;Guldiken et al., 2011;Li et al., 2018;Masoud & Fakharian, 2005;Seddighi & Dehghani, 2002;Taha & Mohammed, 2019;Vega et al., 2017), while in our study, we compared prolactin levels in patients with chronic migraine, episodic migraine in ictal and interictal phases and control subjects separately. In addition to, the correlation between prolactin levels and the number of headache days was assessed in our study. Our findings showed a significant correlation between the number of headache days and serum prolactin levels among EM and CM groups. It could be hypothesized that alterations in prolactin levels may contribute to the activation of headaches in migraineurs and increase the number of headache days that may predispose a person to chronic migraine (Xu et al., 2020) Given the variation in prolactin levels in serum during headache attacks, previous studies have suggested that prolactin may be involved in the mechanism of the attacks (Hering & Kuritzky, 1992;Horrobin, 1973). In addition, hyperprolactinemia in pituitary tumor in which patients often have headaches supports the idea that increased levels of prolactin has been linked to increased migraine attacks (Bosco et al., 2008). In patients with a pituitary tumor, dopaminergic agonists, including cabergoline and bromocriptine, can normalize prolactin levels and decrease headache attacks (Pineyro et al., 2017). The mechanism through which prolactin could affect headache attacks is unknown. Among the neuroendocrinological alterations, hypothalamic-pituitary axis disorders are believed to be engaged in headache pathogenesis (Cavestro et al., 2006 Patil et al., 2013).
Despite these, even normal levels of prolactin could contribute to the sensitization of trigeminal sensory neurons and migraine attacks . However, it is difficult to understand the entire process because of the broad relationships and interaction between all hormones and mediators.
One of the strengths of the present study that sets it apart from other studies is that we evaluated the level of prolactin in the ictal and interictal periods in the EM group and compared the values with those of the CM group as well as with headache-free controls. Further, in this study, EM and CM were accurately diagnosed by an expert headache specialist-neurologist and the results were compared with age-and sex-matched headache-free controls.
Some of the limitations of this study include the case-control design, which could be a source of bias when interpreting the findings because a cause-and-effect relationship cannot be confirmed between migraine and prolactin. In addition, various confounding factors such as stress, which can affect serum prolactin levels, were not considered in our study . Because of the inconsistency of prolactin levels in migraineurs, we suggest further investigation of hormone levels such as dopamine, which is a factor in migraine mechanisms and affects prolactin levels, as well as corticosterone levels, which could provide information on patients' stress levels.

CONCLUSION
In the present study, subjects with chronic and episodic migraine during the ictal phase were shown to have higher serum levels of prolactin than the episodic migraineurs in the interictal phase and age-and sexmatched headache-free individuals. These findings further support the hypothesis that there might be an association between increased prolactin concentrations and the induction of a migraine attack and this may increase the risk for transformation of EM to CM. To determine a possible causal relationship between the prolactin level and pathogenesis and development of migraine headaches or the progression of episodic migraines to chronic migraines, more studies with larger sample sizes should be undertaken.

AUTHOR CONTRIBUTIONS
Study concept and design, patient selection, supervising all steps of the study, and editing the manuscript: M.T. Analysis and interpretation of data and editing the manuscript: Z.G. Drafting of the manuscript: S.H.