An overwhelming majority of the global population, approximately 95%, have suffered from a headache at some point in their lives, with an alarming annual prevalence that suggests nearly half of all adults have experienced a headache within a given year ^[1]. The ramifications of this health issue extend far beyond personal discomfort, with headaches accounting for one-tenth of consultations with general practitioners ^[2], and a significant portion, one-third, of referrals to neurologists ^[3]. Moreover, acute medical admissions related to headaches are alarmingly high, constituting one in every five cases.

The World Health Organization recognizes the debilitating nature of headaches, including them among the top ten global causes of disability. Interestingly, in women, the prevalence and impact of headaches are even more pronounced, ranking among the top five causes of disability ^[4]. It is pertinent to note that the debilitating impact of headaches is comparable to chronic conditions, such as arthritis and diabetes, and its severity exceeds that of conditions like asthma [5,6]^[5][6].

Taking the United Kingdom as an illustrative example, the socioeconomic implications of migraine, a specific type of headache, are vast. An estimated 25 million workdays are lost annually due to migraine, creating an indirect economic burden of nearly GBP 2 billion per year. This figure does not even include the direct healthcare costs associated with managing headaches, such as medication expenses, consultations with general practitioners, referrals to specialists, and visits to emergency care facilities ^[7].

Quantifying the influence of headaches, particularly migraine, on an individual’s quality of life can be challenging. Yet, it is clear from reported data that the impact is substantial. A significant percentage, approximately 75% of patients, experience functional disability during a migraine attack. Additionally, half of the sufferers require the assistance of family members or friends during an attack, causing a significant disruption to their social lives ^[8]. The ripple effect of headaches extends beyond the individuals suffering from them, impacting society at large and requiring serious attention for more effective management strategies ^[9].

In the majority of cases, patients undergo fewer than 15 episodes of headaches per month, a condition identified as episodic migraine. Conversely, there is a subset of individuals who face a more frequent occurrence of headaches—on 15 or more days each month, spanning over three months. Importantly, at least eight of these days should either meet the diagnostic criteria for migraine without the accompanying aura or show responsiveness to treatment specifically designed for migraine. The International Headache Society recognizes this latter classification as chronic migraine [11]^[10]. Chronic migraine, although less common when compared to its episodic counterpart, remains a pervasive and incapacitating issue [12]^[11]. It poses a significant burden on those afflicted with the condition, dramatically impacting their daily lives and well-being [13,14]^[12][13]. This persistent form of migraine continues to be a widespread challenge, necessitating ongoing research and improved therapeutic strategies to ease the strain it puts on sufferers [15]^[14].

2. Definition and Significance of Molecular Markers

Biomarkers, in the realm of medical and biological research, are defined as quantifiable indicators of biological conditions, representing either physical manifestations or results obtained from laboratory tests that correlate with biological processes. These markers have the potential to serve critical diagnostic or prognostic functions [34]^[15]. A more explicit definition of biomarkers was proposed during a conference hosted by the US Food and Drug Administration. In this context, biomarkers are characterized as quantifiable attributes that can be objectively measured and assessed, providing insights into standard biological, pathological, or pharmacological processes [35]^[16]. This clear and precise definition paves the way for a bifurcation of biomarkers into the following two unique types: diagnostic and therapeutic. Diagnostic biomarkers serve as flags for pathological conditions and bear a close association with the risk of developing a disease and its severity. They aid in identifying the presence of a disease and gauging its stage or intensity, thus playing a crucial role in guiding clinical decision-making [36]^[17]. On the other hand, therapeutic biomarkers hold a different but equally important role. They provide information on a treatment’s response, effectively serving as indicators of the efficacy or success of a therapeutic intervention. These biomarkers help clinicians tailor treatments to individual patients, allowing for personalized medicine approaches. They offer a chance to predict whether a patient is likely to respond positively to a particular treatment, making them a powerful tool in the management and treatment of diseases. By providing an early indication of the effectiveness of a therapeutic regimen, these markers can guide healthcare professionals in adjusting treatments as necessary, minimizing the trial-and-error aspect of disease management and increasing the probability of successful outcomes [15]^[14]. Biomarkers represent objective physical traits that can be harnessed to illuminate and distinguish the biological nature and mechanisms of various diseases and syndromes. Essentially, they provide snapshots of the body’s physiological state and can offer valuable insights into health and disease processes. Biomarkers have an extensive range of potential manifestations, which can include but are certainly not limited to, results obtained from the examination of blood, urine, muscle, nerve, skin, or cerebrospinal fluid [37]^[18]. Additionally, biomarkers may also be identified in the form of genes or gene products. These genetic markers offer a unique insight into an individual’s inherent disease susceptibility or resistance and can often illuminate potential therapeutic pathways. Likewise, biomarkers can be identified through advanced imaging techniques such as X-rays, magnetic resonance imaging (MRI), or computed tomographic (CT) scans. These imaging biomarkers can provide a visual representation of disease progression, allowing clinicians to identify anatomical or functional changes in the body over time [34]^[15]. Another fascinating domain of biomarkers lies in the realm of electrophysiological measurements, such as those generated by electrocardiograms (ECGs), electroencephalograms (EEGs), or nerve conduction studies. These types of biomarkers record the electrical activity of the heart, brain, or nerves, respectively, offering a unique insight into the physiological function of these systems. An important issue worth mentioning is those paraclinical investigations offer a new avenue for the management of migraine but are not proven to be of high sensibility and sensitivity for daily physician’s practice. Even though neuroimaging and functional analyses of the brain activity might give a broader point of view regarding therapeutic possibilities, those should not be taken into consideration as absolute clinical criteria. Ultimately, a biomarker could be virtually any characteristic that can be detected, quantified, and expressed in terms of physical qualities. These could include diverse measures, such as height, weight, depth, voltage, luminescence, resistance, viscosity, width, length, volume, or area. Each of these measures contributes to the vast array of biomarkers that hold promise for enhancing our understanding of diseases and guiding the development of effective therapeutic interventions. The utilization of such a wide array of biomarkers allows for a comprehensive, multi-faceted approach to understanding and treating diseases, ultimately leading to more effective and personalized healthcare solutions [38]^[19].

3. Identification of Potential Molecular Markers Associated with Migraine

The National Institutes of Health Biomarkers Definitions Working Group, in 1998, presented a definition for biomarkers. As per their definition, a biomarker refers to “a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention” [35]^[16]. Biomarkers may be classified based on their functional roles, such as diagnostic, therapeutic, risk, progression, and prognostic indicators. The ‘ideal’ biomarker is characterized by the following features [39]^[20]:

• High sensitivity and specificity: this ensures that the biomarker can accurately identify individuals with a specific condition, and also correctly rule out those without the condition;
• High predictive value: the biomarker should be able to accurately forecast the course of the disease, providing valuable insights for disease management;
• Analytical stability: the biomarker should remain consistent over time and across different conditions, thereby ensuring reliable results;
• Easy, cost-effective, and minimally invasive analysis: the method of assessing the biomarker should be simple, economical, and cause minimal discomfort to the patient;
• Repeatability of method: the assessment method should yield consistent results when repeated, thereby ensuring the reliability of the biomarker.

In the context of migraine, however, there are no validated biomarkers due to the absence of substance or genetic variants that are exclusively associated with this condition or the lack of comprehensive studies on potential biomarkers.

3.1. Markers of Inflammation and Oxidative Stress

The markers of inflammation and oxidative stress have been associated with migraine in several studies. Proinflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), have been implicated in this condition [40]^[21]. It has been found that the level of IL-1α is elevated in the blood of children suffering from migraine with aura (MA) [40]^[21]. Similarly, adults with MA have been found to exhibit higher plasma levels of IL-1β during headache-free periods and early stages of attacks as compared to those suffering from migraine without aura (MO) [40,41]^[21][22]. The concentration of IL-6 is reported to increase during the initial two hours of a migraine attack. Additionally, the levels of IL-10 and tumor necrosis factor alpha (TNF-α) are also found to be elevated during these attacks. It is believed that other inflammatory markers associated with vascular dysfunction, such as homocysteine (Hcy) and matrix metalloproteinase-9 (MMP-9), are also elevated in the blood of individuals with migraine [15]^[14]. Elevated serum Hcy concentration has been linked to migraine with aura (MA), and some studies have noted a relationship between increased Hcy levels and higher frequency and severity of migraine; however, these findings are not supported by all research. Hyperhomocysteinemia (elevated Hcy) is hypothesized to initiate migraine with aura attacks through changes in pain threshold [42,43]^[23][24].

3.2. Markers Associated with Pain Transmission and Emotions

Biochemical research has revealed several metabolic irregularities in the synthesis of neuromodulators and neurotransmitters associated with migraine, particularly migraine without aura (MO). Alterations in the metabolic pathway of tyrosine, for example, lead to abnormal production of neurotransmitters like noradrenaline (NE) and dopamine (DA). This process results in an increase in the levels of trace amines, such as tyramine, octopamine, and synephrine. Such changes compromise mitochondrial function and elevate glutamate concentrations within the central nervous system (CNS), as can be seen in Table 1 [43]^[24]. These imbalances in the neurotransmitter and neuromodulator levels within the dopaminergic and noradrenergic synapses of pain pathways could potentially activate the trigeminovascular system (TGVS), causing the release of the calcitonin gene-related peptide (CGRP). This chain of events is believed to directly trigger migraine attacks [44,45]^[25][26]. CGRP plays a key role in transmitting pain signals and promoting inflammation. Its release is stimulated by the activation of TGVS and severe migraine episodes. Infusion of CGRP has been observed to provoke migraine-like attacks in patients with migraine with aura (MA). It has been reported that during inter-attack periods, the saliva and plasma levels of CGRP in migraine patients are significantly higher compared to healthy individuals [43]^[24]. Research conducted on cultured trigeminal neurons suggests that migraine treatment strategies can inhibit CGRP transcription and curtail its release, while tumor necrosis factor alpha (TNF-α) may stimulate the transcription of this peptide [15]^[14]. Another study proposes that high levels of CGRP in saliva may correlate with a significantly improved response to rizatriptan treatment, suggesting that CGRP could serve as a valuable therapeutic marker [46]^[27]. Glutamate, which could potentially activate pathways involving both TGVS and cortical spreading depression (CSD), has been found in elevated concentrations in the plasma, platelets, and cerebrospinal fluid (CSF) of migraine sufferers, including those with chronic migraine. Research suggests that a reduction in plasma glutamate levels could be a marker of a positive response to prophylactic treatment in MO patients [43]^[24]. Serotonin (5-HT) release from platelets into the plasma may be implicated in the pathophysiology of the aura phase of migraine. Izzati-Zade observed a depletion of 5-HT stored in platelets during migraine attacks; moreover, a pattern has been observed in which the plasma level of 5-HT decreases between migraine attacks and the level of the corresponding metabolite, hydroxyindoleacetic acid (5-HIAA), increases. This pattern reverses during migraine attacks [47,48]^[28][29]. This correlation suggests that low 5-HT levels might enable the activation of the trigeminovascular nociceptive pathway triggered by CSD, thus supporting the hypothesis that migraines are a syndrome of low serotonergic disposition. Additionally, a significantly higher concentration of hypocretin-1, a wakefulness-promoting neuropeptide, has been detected in the CSF of patients with chronic migraine, and this has been observed to correlate with painkiller usage [49,50]^[30][31]. Elevated hypocretin-1 levels may be indicative of the early stages of a migraine attack. Conversely, a study involving patients with cluster headaches reported reduced hypocretin-1 levels in the CSF, suggesting that low hypocretin-1 concentrations might reflect insufficient antinociceptive activity in the hypothalamus [51]^[32]. New therapeutic targets for migraine treatment, such as CGRP receptor antagonists, anti-CGRP antibodies, 5-HT1F agonists, glutamate antagonists, and dual hypocretin-1 receptor antagonists, are currently under investigation in phase II clinical trials [52,53]^[33][34]. These emerging therapies reflect the continuous exploration and evolution of our understanding of migraine pathophysiology.

Molecule	Migraine Type (Chronic Migraine [CM]/Episodic Migraine [EM])	Action in Relation to Migraine
Sodium [54,55][35][36]	EM	During a migraine, there is an increase in cerebrospinal fluid (CSF) sodium concentration, while the blood plasma sodium concentration remains unchanged. Additionally, sodium excursions may follow a temporal pattern that worsens migraine in susceptible patients
Homocysteine [56][37]	EM	High levels of homocysteine are potentially linked to migraine with aura and an increased risk of cardiovascular events in patients with migraine
3,4-Dihydroxyphenylacetic acid (DOPAC) [57][38]	EM	Related with dopaminergic activity Positive correlation between the concentration of DOPAC (3,4-dihydroxyphenylacetic acid) and the intensity of migraine, whether with or without aura
Phosphatidylcholine-specific phospholipase C [58][39]	EM	The process involves the hydrolysis of phosphatidylcholine, resulting in the production of important second messengers, diacylglycerol, and phosphorylcholine
Transforming growth factor-β1 [59][40]	EM, CM	An anti-inflammatory cytokine
Interleukin-1 receptor antagonist [59][40]	EM, CM	Proinflammatory cytokine
Monocyte chemoattractant protein-1 [59][40]	EM, CM	Proinflammatory cytokine
Corticotrophin-releasing factor [60][41]	CM, MOH	May be involved in activation of hypocretin/orexin system.
Orexin-A (also referred to as hypocretin-1) [60][41]	CM, MOH	Involved in the maintenance and regulation of various physiological functions, including arousal, sleep, appetite, drinking behavior, central control of autonomic activity, certain endocrine responses, and pain modulation
Glial cell line-derived neurotrophic factor [61][42]	CM	It may play a role in pain relief by regulating the expression of sodium channel subunits, capsaicin VR1 receptors, and substance P release Reduced levels found in patients with migraine
Somatostatin [61][42]	CM	Regulatory anti-inflammatory and antinociceptive peptide
Glutamate [62][43]	CM	The primary excitatory neurotransmitter in the central nervous system. It has been linked to various migraine-related processes, including cortical spreading depression, trigeminovascular activation, and central sensitization.
Tumor necrosis factor-α [63][44]	CM	A proinflammatory cytokine that plays a significant role in brain inflammatory and immune processes, as well as in the initiation of pain
Taurine [64][45]	EM, CM	Inhibitory effect on neuronal activity and vasodilating properties
Glycine [64][45]	EM, CM	Inhibitory neurotransmitter
Glutamine [64][45]	EM, CM	May be involved with initiation and propagation of spreading cortical depression
Neuropeptide Y [65][46]	Acute migraine	Strong vasoconstrictor