Scientific Symposium: Biological Sex as a Factor in Epilepsy Research and Treatment

Abstract

Epilepsy disorders differ depending on sex, gender and hormonal status. This can change treatment efficacy and remains an understudied topic in epilepsy, despite several public health initiatives promoting the inclusion of female subjects in clinical and preclinical research over the last 40 years. Here, we summarize research presented at the 2025 American Epilepsy Meeting Scientific Symposium that described current knowledge in sex differences in epilepsy, including X chromosome-dependent and -independent genetic and epigenetic mechanisms, and discussed how these advances may impact epilepsy management. The importance of considering sex, gender and hormones in epilepsy is increasingly acknowledged in basic, preclinical and clinical research. It is crucial to leverage this momentum into large-scale population research to better characterize the differences alongside detailed mechanistic studies that can ultimately lead to improved care for all individuals with epilepsy.

Introduction

Epilepsy disorders can display sex and gender differences and are affected by gonadal hormones.^1,2 It is thus likely that, to be most effective, therapies must be tailored based on biological sex and hormonal status. More than 30 years ago, in 1985, the U.S. Public Health Service Task Force acknowledged that the exclusion of women from clinical research was detrimental to their health. ³ Eight years later, the NIH Revitalization Act mandated that women and minorities be included in all NIH-funded clinical research and that Phase III clinical trials be analyzed for sex differences. ⁴ In 2015, the National Institutes of Health required the use of male and female subjects in all federally funded research, preclinical and clinical. Despite these initiatives to increase research including men and women, there is still a considerable knowledge gap in sex-specific aspects of epilepsy. Sex differences in epilepsy come in many flavors spanning molecular, cellular and brain physiological levels. Perhaps unsurprisingly, X chromosome-linked epilepsy syndromes differ in severity and phenotypic presentation between male and female individuals. However, more recent evidence suggests that even X chromosome-independent epigenetic mechanisms, such as histone modification or regulation by noncoding RNAs, differ in females affecting epilepsy phenotypes and the efficacy of experimental treatments. In addition, ovarian hormones can alter seizure susceptibility, as seen in catamenial epilepsy, in which seizure frequency varies across the menstrual cycle. Conversely, chronic seizures can modify the menstrual cycle, leading to a reciprocal relationship between the female reproductive cycle and epilepsy with a prominent role of the hypothalamic–pituitary–gonadal (HPG) axis, which links the brain and reproductive endocrine systems in all mammals. This short review, based on a scientific symposium held at the 2025 American Epilepsy Society Meeting, touches on topics across the entire breadth of sex-related issues in epilepsy from genetic and epigenetic to hormonal mechanisms and discusses the implications of biological sex for epilepsy management. There is increasing awareness for sex differences in epilepsy. Here, we emphasize the importance of translating this momentum into actionable knowledge to improve treatment.

Genetic Mechanisms in Sex Differences: X-Chromosome Inactivation in Epilepsy

X-linked epilepsies are neurologic disorders caused by mutations in X chromosome genes, with seizures as a primary feature (reviewed in Bernardo et al ⁵ ). These disorders highlight the intersection of genetics, cellular biology, and sex-specific mechanisms, notably involving X-chromosome inactivation (XCI), inheritance patterns, mosaicism, and cellular interference.

XCI is a process in females where one X chromosome is randomly silenced early in development, producing a mosaic of cells expressing either maternal or paternal X-linked genes. ⁶ Females with X-linked mutations have a mix of normal and mutated cells, while males, possessing a single X chromosome, are uniform in gene expression. Skewed XCI, where one X chromosome is preferentially silenced, can significantly alter disease expression in female carriers.

In X-linked dominant disorders, both sexes may be affected if they carry the mutation, while in X-linked recessive conditions, males are typically affected, and females show phenotypic variability due to random/skewed XCI. A unique inheritance pattern occurs in PCDH19 clustering-epilepsy (PCE), termed “cellular interference.” Here, symptoms are often more severe in females due to interactions among genetically distinct cell populations. ⁷

PCE results from pathogenic mutations in PCDH19, a gene critical for neuronal migration, circuit formation, and synaptic maintenance. ⁸ PCE affects predominantly females (and rare mosaic males), causing clusters of difficult-to-treat seizures in early childhood, sometimes alongside cognitive disabilities, autism, and psychiatric symptoms. ⁹ Notably, hemizygous males do not typically present with epilepsy, emphasizing the importance of mosaicism due to XCI in females. Most PCE patients are reported to have a normal brain MRI, though subtle cortical malformations are increasingly reported. ¹⁰ Advanced morphometric analyses across affected individuals highlight correlations between regional PCDH19 gene expression and decreased cortical surface area. More severe neuropsychiatric presentations correspond with greater alterations in regions such as the hippocampus and entorhinal cortex. ¹¹

Laboratory modeling has yielded significant insight into PCE pathogenesis, using mouse, zebrafish, and human cortical organoid models.^12–15 Notably, these studies reveal unique cell segregation patterns in the cortex and hippocampus; cells expressing wild-type or mutant Pcdh19 often cluster in a “striping” pattern which varies between individuals.^13,14 Additional modeling has suggested that Pcdh19 mosaicism alters interneuron migration and GABAergic signaling, disrupting interneuron distribution. ¹⁶ Indeed, recent mouse modeling of PCE has shown that PCE mice have a decreased seizure threshold to a physiologically relevant hyperthermia assay, and that there are allele-specific alterations in the density of parvalbumin-positive inhibitory neurons. ¹⁷

Collectively, these findings support that PCDH19 mosaicism can affect the cellular composition and structure of focal brain regions, contributing to the diverse symptoms of PCE. Overall, X-linked epilepsies like PCE demonstrate the complex interplay of genetic inheritance, X-inactivation, and cellular biology, influencing not just who develops epilepsy, but also its cellular and clinical manifestation. Understanding these mechanisms is crucial for counseling affected individuals and developing targeted therapies for X-linked epilepsies.

Epigenetic Mechanisms in Sex Differences: Chromatin Modifiers in Epilepsy

Brain phenotypes arise from interactions between genetic factors and environmental influences. In epilepsy, observed sex differences highlight the importance of X-chromosome genes as sexually dimorphic risk factors for brain disorders. The neural environment is also shaped by systemic physiological signals, including circulating sex hormones, which regulate neuronal function through receptor-mediated pathways. Epigenetic modifiers provide a mechanistic link between X-linked genetic risk and hormonal influences by regulating gene expression without altering DNA sequence. These mechanisms mediate environmental signals, including those from sex steroids, and contribute to sex-specific gene expression in neurons.

Epigenetic mechanisms are central to biological sex (reviewed in Kundakovic and Tickerhoof. ¹⁸ ). The three major epigenetic mechanisms are DNA methylation, histone modification, and regulation by noncoding RNAs (reviewed in Zhang et al ¹⁹ ). DNA methylation involves the addition of methyl groups to CpG sites, typically in gene promoter regions, leading to transcriptional repression. Histone modifications regulate how tightly DNA is packaged around histone proteins through reversible chemical changes such as methylation, acetylation, and phosphorylation, thereby influencing gene accessibility. Regulation by noncoding RNAs provides an additional layer of epigenetic control and plays a role in sex differences in epilepsy (discussed below). Together, these mechanisms shape molecular pathways underlying biological sex differences.

A key link between epigenetics and the X chromosome is XCI, which is mediated by the long noncoding RNA Xist. ²⁰ Xist is transcribed from the future inactive X chromosome and coats it, serving as a scaffold that recruits chromatin-modifying complexes. ²⁰ These include Polycomb Repressive Complex 1 (PRC1) and PRC 2 (PRC2), which deposit repressive histone marks and remove active chromatin modifications. Components of these complexes, including EZH2 (the catalytic subunit of PRC2), RNF2 (PRC1), and MYBBP1A (a PRC2-associated protein), have been linked to neurodevelopmental disorders and epilepsy (reviewed in Bölicke and Albert. ²¹ ). Importantly, pathways opposing PRC2 activity are responsive to sex steroids, and PRC2 subunits can be regulated through direct interactions with estrogen receptors,^22,23 suggesting potential sex-specific effects of disruption in these pathways. These and other epigenetic proteins, including Absent, small, or homeotic-like 1 (ASH1L), are encoded by genes that cause a large number of neural developmental disorders. ²⁴

The ASH1L gene encodes a histone methyltransferase that antagonizes PRC2-mediated transcriptional repression.^25,26 ASH1L is a strong genetic risk factor for autism spectrum disorder (ASD), and haploinsufficiency is associated with a neurocognitive profile including, but not limited to ASD, intellectual disability, and epilepsy. ²⁷ Although reported cases predominantly involve affected males, ²⁷ our preliminary study ²⁸ suggests sex-specific phenotypes: males show ASD with mild or absent epilepsy, whereas females exhibit epilepsy with lower ASD symptom severity. Mouse studies support sex-specific circuit effects, with increased hippocampal excitability in females but not males, while other brain regions did not demonstrate these differences. Whether these sex-specific differences arise from altered XCI, unopposed PRC2 activity, or interactions with sex hormones remains an important area for future research.

Hormonal Mechanisms of Sex Differences: Reciprocal Loops Link Epilepsy and the Reproductive Neuroendocrine Axis

Clinical evidence documents sex-influenced impacts of epilepsy on the HPG axis, with women and men with epilepsy showing increased rates of multiple reproductive endocrine disorders (for a recent comprehensive review, please see Cutia and Christian-Hinman ²⁹ ), including changed luteinizing hormone (LH) and follicle-stimulating hormone levels and release patterns. At the gonadal level, altered estrogen and/or progesterone levels and hyperandrogenism are commonly found in women with epilepsy, whereas men with epilepsy are at increased risk of low testosterone, even when accounting for endocrine side effects of antiseizure medications (ASMs).

From the preclinical perspective, female rats and mice that develop chronic epilepsy after electrical kindling of the amygdala, intrahippocampal kainic acid (IHKA) injection, or systemic pilocarpine-induced status epilepticus can demonstrate irregular estrous cycles, changes in pituitary and gonadal hormone levels, and ovarian cysts.^30–32 Increased serum testosterone and testis weight were found in male rats with epilepsy after amygdalar kindling, ³³ but not in IHKA mice. ³⁴

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons from IHKA mice with chronic epilepsy show changes in action potential firing, excitability, and voltage-dependent potassium currents, with effects shaped by sex, estrous cycle stage, and cycle regularity.^34–36 Notably, depolarizing GABA input to GnRH neurons appears to be specifically elevated only in IHKA females with disrupted estrous cycles. ³⁷ At the pituitary level, IHKA females, but not males, exhibit altered gene expression and increased LH release in response to peripheral GnRH injection, and this effect is observed on diestrus but not estrus. ³⁸

Reciprocally, gonadal hormone signaling exerts sex-dependent modulation of neural excitability and seizures. As further discussed below, higher estradiol:progesterone ratios midway through the menstrual cycle are thought to drive periovulatory seizure exacerbations in catamenial epilepsy, whereas the drop in progesterone and consequent reduction in progesterone-derived neurosteroid metabolites at the end of the cycle promote perimenstrual seizures. ³⁹ The effects of gonadal hormones on seizures have received far more attention in women, but the use of letrozole, an aromatase inhibitor that prevents the conversion of testosterone to estradiol in the brain, may have antiseizure benefits in men. ⁴⁰

Preclinical rodent models of epilepsy also show sex-specific gonadal hormone effects on seizure induction (for review, see Christian et al ⁴¹ ). Seizure susceptibility, interictal spiking, and chronic epilepsy burden can shift with estrous cycle stage in female rodents. The effects of estradiol in female rodents are primarily, although not exclusively, proconvulsant. Similarly, progesterone-derived neurosteroids reduce excitability and seizures, but nuclear progesterone receptor activation can elevate excitability. In both sexes, testosterone has multiple effects on seizures through conversion to estradiol, dihydrotestosterone, or neurosteroids such as 3α-androstanediol.

Several preclinical studies have documented sex differences in both acute seizure induction and chronic epilepsy. ⁴¹ For example, a recent study in IHKA mice showed more severe epilepsy in females and an influence of hippocampal seizure lateralization that was not observed in males. ⁴² Another recent report showed sex differences in acute behavioral seizure susceptibility and mortality that were “unmasked” by gonadectomy, ⁴³ further emphasizing how interactions between sex and gonadal hormone signaling can shape seizure outcomes.

The Role of Noncoding RNAs in Sex Differences: Implications for Targeted Therapy Development in Epilepsy

MicroRNAs are short noncoding RNAs that regulate the stability and translation of many different mRNAs. MicroRNAs mostly act as negative regulators of gene expression, and one microRNA usually can affect the expression of several hundred mRNAs. ⁴⁴ This makes them promising therapeutic candidates that could correct the extensive changes in gene expression after seizures and in epilepsy. It is thus not surprising that microRNAs have been implicated as potential treatment targets in preclinical studies ⁴⁵ and suggested as blood or CSF biomarkers for epileptogenesis or drug resistant epilepsy.

As is the case with many epilepsy studies, this research was mainly done in male subjects without considering potential sex differences. Therefore, knowledge about sex differences in microRNA regulation in the context of epilepsy is scarce. A recent study analyzing the proconvulsant microRNA miR-324-5p in female mice discovered important sex differences that may affect miR-324-5p as epilepsy treatment target in females. Studies in male mice showed that antisense-mediated inhibition of miR-324-5p increases the latency to seizure onset after a proconvulsant stimulus in male mice and reduces seizure frequency in a mouse model of acquired epilepsy.^46,47 However, miR-324-5p inhibition is not effective in reducing kainic acid-induced seizure susceptibility in female mice. ⁴⁸ Moreover, seizures affected the activity of miR-324-5p differentially in males compared with female mice: whereas in male mice, seizures lead to increased recruitment of miR-324-5p to the RNA-induced silencing complex (RISC) and thus increased activity, the opposite is observed in female mice, potentially caused by ovarian hormones.

Given the paucity of research in this area, it is highly likely that these findings are just the tip of the iceberg of sex-dependent regulation of microRNAs in epilepsy. Surprisingly little is known about the effects of sex and gonadal hormones on microRNA function in the brain. For example, it is unknown if or how the RISC is affected by biological sex, and if microRNA activity differs in the brains of male and female subjects, or across the menstrual cycle. Of note, the X chromosome is enriched in microRNAs and pilot studies have associated several of these microRNAs with X-linked intellectual disability. ⁴⁹ Recent microRNA profiling studies have identified sex-biased expression of more than 20% of all known microRNAs in the developing mouse brain, of which less than 10% were located on the X chromosome.^50,51 Initial analyses suggested that estrogen receptor subunits differentially affect transcription of sex-biased pre-microRNAs. ⁵⁰

The results discussed above highlight that both sex and gonadal hormones influence microRNA activity in the brain, can affect brain function, and underlie sex-specific epilepsy disorders. These novel studies paint an incomplete picture of sex-specific regulation of microRNAs but suggest they play an important role in sex differences in the brain. Interestingly, postmortem studies in patients after stroke revealed both distinct and overlapping microRNAs in male and female patients, ⁵² suggesting that similar phenomena could be present in epilepsy. Given the potential that epigenetic regulators such as microRNAs have as treatment targets in complex network disorders like epilepsy, future studies are urgently needed to further delineate how sex and hormones affect microRNAs in the brain and contribute to male- or female-specific epilepsy disorders.

Implications of Biological Sex for Epilepsy Management

Biological sex and sex steroid hormones (SSH) have profound implications for epilepsy pathophysiology, treatment response, and clinical management across the lifespan. Personalized care must account for endogenous SSH changes as well as exogenous hormonal therapies, which can influence seizure susceptibility and ASM pharmacology.

Hormonal effects on epilepsy emerge early. At puberty, some epilepsies remit (eg, childhood absence epilepsy), while others begin (eg, juvenile epilepsies). During the reproductive years, predictable cyclical fluctuations in estradiol and progesterone may parallel predictable patterns of seizure exacerbation, termed catamenial epilepsy. Three clinically useful patterns are recognized ⁵³ : perimenstrual (C1), periovulatory (C2), and anovulatory cycles (C3), together affecting approximately half of women with epilepsy across independent cohorts.^53,54 Despite long-standing interest in hormonal therapies, few high-quality interventional studies exist. In the NIH progesterone trial, cyclic progesterone supplementation was not superior to placebo overall. ⁵⁵ This result may reflect the complex biology of progesterone, both anticonvulsant via GABAergic neuroactive metabolites (eg, allopregnanolone) and potentially proconvulsant through nuclear receptor–mediated upregulation of AMPA receptor subunits. Importantly, subgroup analyses demonstrated that women with a strong perimenstrual (C1) pattern had significantly higher responder rates to progesterone, with seizure reduction correlating with increases in allopregnanolone. ⁵⁶ These findings emphasize the importance of patient selection based on hormonal–seizure coupling. However, existing trials of synthetic allopregnanolone analogs were not designed to test the effects in this patient population. Also, despite increasingly diverse contraceptive methods, evidence largely focuses on reduced efficacy with enzyme-inducing ASMs, while their effect on seizure susceptibility remains inadequately studied.

Pregnancy represents an extreme hormonal state, with SSH concentrations increasing up to 50-fold and rapidly declining postpartum. These changes alter ASM pharmacokinetics, increasing clearance of most ASMs, making therapeutic drug monitoring (TDM) essential. ⁵⁶ When TDM is applied, overall seizure control is often maintained. Nonetheless, seizure worsening during pregnancy is associated with lower third-trimester allopregnanolone concentrations ⁵⁷ and disproportionately affects women with focal (particularly frontal lobe) epilepsy, ⁵⁸ those on polytherapy, and those without preconception seizure freedom—factors pointing to distinct biological and clinical vulnerability profiles.

Later in life, the menopausal transition introduces further complexity, with less predictable and poorly characterized hormonal fluctuations. Women with epilepsy are at increased risk of premature ovarian failure; ⁵⁹ however, as with fertility and polycystic ovarian syndrome, the respective roles of seizures and ASMs are poorly understood. Seizure frequency may worsen during perimenopause before stabilizing or improving after menopause. One randomized trial using a combination of conjugated estrogens and medroxyprogesterone showed a dose-dependent increase in seizure frequency but also reduced lamotrigine levels. ⁶⁰ Contemporary menopause guidelines emphasize individualized risk–benefit assessment, raising the possibility that selected menopausal hormone therapies could stabilize hormonal variability and benefit certain patients both in terms of seizures and comorbidities.

Across all life stages, major gaps remain: how best to biologically define catamenial epilepsy; whether biomarkers such as neuroactive steroids can guide therapy; how SSH interact with seizure networks and ASM metabolism; and which patients may benefit from targeted hormonal/neurosteroid supplementation. Until these questions are answered, optimal epilepsy care in women requires vigilant ASM optimization, thoughtful integration of hormonal strategies, and anticipatory monitoring across reproductive transitions.

Conclusion and Future Outlook

Sex differences in epilepsy are multifaceted and wide-ranging. It has long been known that gonadal hormones can alter seizure susceptibility, and vice versa, epilepsy can change the reproductive cycle. The shift in NIH funding strategy to require inclusion of male and female subjects in basic research about 10 years ago has raised awareness and increased the number of studies incorporating sex as a biological variable. This research has led to insight into X chromosomal, epigenetic and gonadal hormone-related mechanisms contributing to sex differences in neuronal excitability and susceptibility to seizures. Despite these advances, we are still far from fully understanding the impact of sex and gonadal hormones on epilepsy severity and outcomes, and most importantly, their effects on the efficacy of therapeutic treatments. More preclinical research into underlying mechanisms as well as large scale human studies to better understand the impact of sex on epilepsy are needed to improve epilepsy care and treatment across the lifespan of both male and female patients, and to leverage sex differences into novel more effective treatments.