The BBB is composed of brain endothelial cells that create a continuous, non-fenestrated microvascular network in the central nervous system (CNS) (Cho et al., 1999). A detailed review of the structure and function of the BBB was recently given by Zhang et al. (Zhang et al., 2022). In short, tight junctions, adherens junctions, and gap junctions between endothelial cells control the movement of ions, molecules, and cells from the blood to the brain parenchyma (Hawkins et al., 2006). Tight junctions are created by homotypic and heterotypic interactions between transmembrane proteins including claudins, occludins, and junction adhesion molecules (JAMs), that connect to the actin cytoskeleton via cytoplasmic scaffold proteins of the membrane-associated guanylate kinase (MAGUK) family (Yuan et al., 2020; Hashimoto et al., 2023; Sugiyama et al., 2023). Conversely, adherens junctions are formed by members of the cadherin protein family and are linked to the actin cytoskeleton by catenins (Weis and Nelson, 2006). A thin sheet of basement membrane (BM) composed of glycosaminoglycans, proteoglycans, and glycoproteins surrounds the endothelial barrier and provides structural support to the neurovascular network (Tewari et al., 2022). The composition of the brain extracellular matrix (ECM) changes with age and disease, which has effects on the integrity of the BBB (Thomsen et al., 2017); ECM composition as well as biomechanical properties have been reported to vary by sex (Batzdorf et al., 2022). Surrounding the BM are pericytes, astrocytes, and microglia that secrete ECM molecules and other signals to influence the BBB (Bisht et al., 2021; Kisler et a., 2021; Gao et al., 2023). Together, these glial cells offer homeostasis and structural support to the BBB, and any deviation can result in disease (Tewari et al., 2022). Pericytes reside within the BM and communicate with endothelial cells and astrocytes through the secretion of signals (Armulik et al., 2010; Wu et al., 2023). Astrocytes extend end foot processes into the BM, where they play major roles in the clearance of waste, regulating BBB permeability, and modulating synaptic transmission (Abbott et al., 2006; Tewari et al., 2022; Wu et al., 2023). Microglia are the brain’s resident immune cells that are responsible for innate and adaptive immune responses. These glial cells, along with the endothelium and local neurons, are termed the neurovascular unit (Zlokovic, 2008; Sato et al., 2022).

Sex-related differences in the behavior of glial cells in non-disease states have previously been reported. An in-depth review conducted by Lenz and McCarthy describes the various roles microglia play in brain development, including regulation of neural stem cell populations, synaptogenesis, and sexual determination (Lenz and McCarthy, 2015). Microglia in multiple regions of the developing rodent brain show sex differences in colonization, gene expression, and cytokine secretion (Schwarz et al., 2012). Sex differences in microglia phenotypes also extend into adulthood in animal models and are retained independently from the in vivo steroid hormone profile (Villa et al., 2018; Doran et al., 2019). These observed differences in phenotype establish that male and female microglia behave distinctly. Additional studies are needed to determine the extent that sex-related differences in microglia phenotypes impact the BBB. Similarly, sex steroid hormones influence astrocyte function and morphology. Studies have shown that estradiol is implicated in the expansion of astrocyte processes and regulation of intracellular ion concentration (Acaz-Fonseca et al., 2014). Importantly, this may have profound sex-dependent effects on the communication between astrocytes and other cells of the BBB, which is yet to be explored. Further research is required to elucidate sex-dependent function of the neurovascular unit under normal physiological conditions.

The permeability of the BBB is tightly regulated by glial cells, which work together to sense the environment and produce signals to alter the flow of materials from the blood to the brain tissue. In the context of disease or injury, the normal signaling between glial cells and the brain endothelium is disrupted, resulting in a dysfunctional BBB (Xing et al., 2012). Microglia become activated when surface receptors come in contact with damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs), which leads to the activation of pro-inflammatory machinery and induced activation of surrounding microglia (Lawrence et al., 2023). Activated microglia phagocytose cellular debris caused by the injury or disease, leading to the release of signals such as tumor necrosis factor-α (TNF-α) that act on local astrocytes. The subsequent change in gene expression and phenotype of astrocytes in response to the local environment is termed reactive astrogliosis. Reactive astrocytes promote pro-inflammatory conditions by secreting complement components and chemokines that attract circulating peripheral immune cells (Liddelow and Barres, 2017). These disease processes exacerbate oxidative stress and BBB dysfunction through the generation of pro-inflammatory signals, free radicals, and matrix metalloproteinases (MMPs) (Zhao et al., 2022). Secretion of pro-inflammatory cytokines, such as IL-1B and TNF-α, degrade tight junction proteins via multiple signaling pathways. Overproduction of free radicals by microglia and astrocytes, along with any inherent disease-induced free radical accumulation, leads to cellular damage and further disruption of the BBB. In parallel, the secretion of MMPs intensifies BBB breakdown through the direct digestion of tight junction and adherens junction proteins. In summary, the pathophysiology of the injured BBB is complex and involves all glial subtypes. Adding to this complexity, previous literature has illuminated sex-related and hormone-related differences in glial cell response to pathological conditions. A recent study of female primary cortical astrocytes reveals estradiol and 5ɑ-androstane-3β,17β-diol (a dihydrotestosterone metabolite) may offer protection against cytotoxic challenge with iodoacetic acid (Kim et al., 2023, p. 3). The authors speculate that the protective effects may result from steroid induced inhibition of connexin 43 gap junction opening. Specifically, this channel is important for cellular signaling and changes in channel permeability have been associated with disease states. Estradiol has also previously been implicated in pathways involved in pericyte attachment and migration in response to inflammatory stimuli (Kurmann et al., 2021). Kurmann and others show estradiol treatment prevents cultured human brain vascular pericyte migration through estrogen receptors ER-ɑ and ER-β signaling. Furthermore, their transcriptomics analysis reveals that estradiol treatment induces changes in expression of transcripts associated with cell migration under inflammatory conditions. Since astrocytes and pericytes are involved in maintaining BBB integrity, these findings suggest a critical role of hormone-mediated protection in the context of brain injury. Additionally, previous studies reveal ex vivo cultures of male neonatal rat microglia stimulated with lipopolysaccharide (LPS) have increased pro-inflammatory responses compared to female cultures (Loram et al., 2012). This leads into the next section, which discusses how biological sex plays a role in peripheral immune responses to brain injury.

Neutrophils are one of the first peripheral immune cells to reach the CNS and may persist chronically after injury (Alam et al., 2020; Mohamud Yusuf et al., 2022). Despite their initial avail, neutrophils are implicated in pathogenic processes associated with chronic neuroinflammation, such as hyperpermeability of the BBB, edema, and reactive oxygen species (ROS) metabolism (Liu et al., 2018). Notably, recent studies have revealed sex differences in neutrophil activation, function, and apoptosis. A multi-omic analysis of mouse neutrophils revealed sex differences in chromatin architecture, with male cells exhibiting increased chromatin compaction compared to female cells (Lu et al., 2021). Chromatin architecture is an important aspect of neutrophil biology due to its involvement in neutrophil extracellular traps (NETs), and the observed sex differences in chromatin architecture may contribute to sex-specific neutrophil responses and gene expression. Another study of isolated human neutrophils revealed that cells obtained from male subjects exhibit higher expression of genes associated with immature phenotypes compared to female cells (Gupta et al., 2020). Neutrophils isolated from healthy females show increased activation, as evident by female-specific gene set enrichment of pathways related to type I interferon signaling and stimulation in the absence of stimulus. It was also noted that male neutrophils treated with estradiol show similar mitochondrial metabolism compared to untreated female neutrophils, which suggests that differences in steroid hormone profile, rather than X chromosome dosage, lead to sex-specific neutrophil phenotypes. To test this hypothesis, researchers analyzed neutrophil type I interferon signaling/stimulated genes in males with Klinefelter syndrome (XXY) and prepubescent volunteers. Female (XX) neutrophils consistently exhibited higher expression of type I interferon signaling/stimulated genes compared to XY and XXY males, and no significant statistical differences in expression between the XY males and XXY males. Additionally, there were no significant differences in the maturation profile or expression of type I interferon signaling/stimulated genes of prepubescent male and female neutrophils. Together these findings suggest that sex steroid hormones contribute to determining female-specific neutrophil physiology in healthy individuals. There are also reports of sex-specific neutrophil responses to pro-inflammatory signals. For example, Pace and others demonstrated that neutrophils isolated from human male subjects exhibit increased production of prostaglandin-E2 (PGE2) upon stimulation with lipopolysaccharide compared to those isolated from female subjects (Pace et al., 2017). PGE2 is a bioactive molecule involved in homeostatic and inflammatory processes. The authors hypothesize this may be due to sex differences observed in COX2 expression, as this is the key enzyme involved in prostaglandin biosynthesis. Ultimately, these findings show there are sex differences in neutrophil biology, and additional research is needed to understand the extent of these effects. Given that neutrophils play a crucial role in inflammation after brain injury, sex differences in neutrophil function may contribute to the sex differences in CNS pathology.

Monocytes, like neutrophils, infiltrate the CNS rapidly in the event of an injury in response to chemoattractants (Jin et al., 2012; Alam et al., 2020). Once monocytes have entered the brain tissue they mature into tissue resident macrophages and begin to perform effector functions including production of inflammatory mediators and phagocytosis of cellular debris (Yang et al., 2014; Alam et al., 2020). Recent studies have revealed hormone-related and sex-related differences in macrophage development and characteristics. Consiglio and others show androgen receptor signaling plays a role in monocyte development in male mice (Consiglio and Gollnick, 2020). They found that deletion of androgen receptors in myeloid cells leads to reduced numbers of mature monocytes and increased numbers of macrophages in bone marrow compared to controls, suggesting androgen receptor signaling increases monocyte development. Additionally, a study of isolated peritoneal macrophages from young and middle-aged rats revealed young females exhibit an increased percentage of macrophages that express activation markers toll-like receptor 4 (TLR4) and major histocompatibility complex II (MHCII) compared to young males (Ćuruvija et al., 2017). It was also reported that when stimulated with lipopolysaccharide (LPS), isolated macrophages from young female rats produced higher levels of interleukin 6 (IL-6) and interleukin 1β (IL-1β) compared to young male macrophages. These findings highlight the impact of sex and age on macrophage phenotypes and response to inflammatory stimuli in animal models. Future studies should focus on elucidating sex-related and hormone-related macrophage responses to better understand the roles they may play in CNS injury. The next section will review reported sex differences in traumatic brain injury (TBI) and stroke.

As one of the leading causes of injury-related death and disability, TBI, affects between 50 and 60 million people globally each year (Maas et al., 2022). The global economic burden of the disease is estimated to be $400 billion per year. Previous literature reveals males have higher rates of TBI compared to women (Mikolic et al., 2021; Centers for Disease Control and Prevention, 2021; Centers for Disease Control and Prevention, 2022). In 2016 and 2017, males were reported to have higher rates of TBI-related hospitalizations for all injury mechanisms including unintentional falls, motor vehicle crashes, and assault (Centers for Disease Control and Prevention, 2021). Additionally, males were reported to have more than three times higher rates of TBI-related deaths compared to females in 2018 and 2019 (Centers for Disease Control and Prevention, 2022). Despite previous efforts to uncover sex differences in TBI outcomes in preclinical and clinical studies, this topic is still widely considered to be controversial. Remarkably, an extensive review by Gupte and others unveiled that human studies report females predominately experience poorer clinical outcomes than males following TBI (Gupte et al., 2019). They found that study parameters such as study size, stratification of TBI severity, and type of outcome measured have an impact on the extent of sex differences reported in human studies. Furthermore, similar trends extend to preclinical TBI models where the mechanism of injury, sample size, and type of outcome measured appear to have a considerable impact on reports of sex differences. Additional research in both preclinical and clinical domains is needed to decipher the etiology and magnitude of sex differences in TBI.

TBI is characterized by a blow, jolt, or penetration to the head that results in the disruption of brain function, leading to complex pathological processes. The acute injury phase is a direct result of the initial injury, which includes damage to neurons, glia and blood vessels. Following the acute phase is a heterogenous, secondary injury phase that encompasses neuroinflammation, heightened intracranial pressure, dysfunction of the BBB, and neuronal excitotoxicity. Neuroinflammation begins with the activation of microglia, which secrete cytokines and chemokines to attract other immune cells such as neutrophils, monocytes, and lymphocytes, to the injury penumbra (Simon et al., 2017). While the initial inflammation aids in neuroregeneration and the clearance of cellular debris, chronic inflammation in the brain can lead to neuronal cell death and further neurodegeneration (Ziebell and Morganti-Kossmann, 2010; Simon et al., 2017). Transport of DAMPs to peripheral immune organs via the glymphatic system leads to systemic immune responses. For example, preclinical studies of TBI have revealed short-term increases in myeloid cell differentiation in the bone marrow and chronic changes in circulating immune cell profiles and behaviors (Ritzel et al., 2018). Additionally, examination of the thymus has revealed chronic disturbances in T cell maturation after TBI.

Recent preclinical studies have revealed sex differences in TBI pathology. Our group previously assessed macromolecule accumulation profiles in male and female mice after experimental TBI (Bharadwaj et al., 2020). Here, horseradish peroxidase (HRP) staining revealed females experience increased macromolecule accumulation in the injured cortex 24 h post-injury compared to males, suggesting sex-dependent BBB permeability after TBI. Additionally, Schwab and others found that female mice exhibit increased DNA damage compared to males after repetitive mild TBI (Schwab et al., 2022). Female mice exhibited increases in R-loops and oxidative base damage compared to sham levels, which was not evident when comparing sham males to injured males. However, when markers for cellular senescence were examined, males and females expressed comparable increases in p21 and p16 proteins compared to their sham counterparts. The authors stated this suggests that males might be more sensitive to genotoxic stress compared to females, and additional research is needed to elucidate the mechanisms that contribute to injury-induced sex-dependent DNA damage. Furthermore, Villapol and others also report that male mice show increased microglial activation, macrophage infiltration, and cell death compared to females at acute timepoints after moderate CCI (Villapol et al., 2017). In this study biological sex and time post-injury were reported to influence cytokine production in microglia and macrophages up to 30 days post-injury. For example, male mice exhibited increased TNF expression at 3 days post-injury compared to females, whereas females exhibited increased TNF and IL-1 expression at 4 h post-injury. Similarly, a study conducted by Doran and others also reported male mice exhibited increased myeloid cell infiltration at 1 day post-injury and increased microglia count at 3 days post-injury compared to females (Doran et al., 2019). Moreover, sex differences have been reported in clinical studies. Wagner and others evaluated sex differences in CSF glutamate concentration and lactate-pyruvate ratio after severe TBI in adults ages 16–65 years (Wagner et al., 2005). Here, females had higher lactate-pyruvate ratios, indicating females experience increased oxidative stress after severe TBI compared to males. They also report sex associations with 24-h glutamate concentration using multivariate analysis, indicating sex differences in glutamate excitotoxicity post-injury. Both findings provide evidence for sex differences after injury that directly affect cells of the BBB.

Stroke affects approximately 12 million people worldwide each year (Feigin et al., 2021). Ischemic stroke (IS) accounted for about 60% of those cases and hemorrhagic stroke (HS) accounted for the remaining 40%. Notably, in 2019, stroke was the fifth leading cause of death for men and the third leading cause of death for women (Rexrode et al., 2022). Overall, women are considered to have a higher lifetime risk for stroke compared to men. However, age impacts the sex-related risk of stroke, with women having a higher incidence up to 30 years of age and men having a higher incidence thereafter until about age 80 (Vyas et al., 2021). There are also differences associated with the type of stroke experienced by men and women, with women having higher prevalence of subarachnoid hemorrhage and men having higher prevalence of hemorrhagic stroke (Rexrode et al., 2022). Previous studies have revealed sex-related differences in risk factors of stroke. For example, women diagnosed with diabetes have higher risk of stroke compared to men with diabetes (Peters et al., 2014). Some other factors that show sex associations include obesity, hypertension, atrial fibrillation (Vyas et al., 2021). Furthermore, there are female-specific risk factors for stroke including the use of hormone contraceptives and therapies, adverse pregnancy outcomes, and age of menopause onset. These phenomena may be due to sex and age-dependent alterations in circulating steroid hormone profiles and future investigations should lay emphasis on elucidating the causes of the sex-dependent risk of stroke.

IS results from reduced blood flow that disturbs the normal function of the brain, causing neurological deficits. The most common causes of IS include arterial occlusion and venous infarction. Similar to IS, HS results in reduced blood flow to the brain caused by rupture of a cerebral artery rather than occlusion. Ruptures can occur in the cerebrum or in the subarachnoid space (SAS). In cases of HS, intracranial pressure rises quickly as the hemorrhagic blood moves into the SAS, ventricles, and parenchyma (Lauzier et al., 2023). At the core of the ischemic event and downstream from the ruptured vessel, the decrease in blood flow contributes to sustained ischemic conditions leading to neuronal death and secondary injury sequelae (Arai et al., 2011). Conversely, in the surrounding penumbra, other perfusing blood vessels provide some reprieve, resulting in a much slower process of cell death. However, in more severe cases, global ischemia can occur, which causes severe oxygen deprivation to large regions of the brain (Lauzier et al., 2023). In the absence of oxygen, neurons lose the ability to generate ATP and extracellular glutamate quickly accumulates causing excitotoxicity (Arai et al., 2011; Long et al., 2023). Additionally, mitochondrial damage leads to the release of free radicals causing oxidative stress and further damage to surrounding cells (Tian et al., 2022). Resident microglia play dual roles by producing pro-inflammatory cytokines and MMPs that cause further BBB and cellular damage, while also producing growth factors that aid in neuroprotection (Jayaraj et al., 2019). Studies of the consequences of stroke have revealed both acute and chronic systemic immune responses. The disruption of the blood brain barrier after stroke allows DAMPS and cytokines to enter the bloodstream, which leads to systemic immune responses. Increases in blood plasma levels of pro-inflammatory cytokines occur as early as 4 h after experimental ischemic stroke (Offner et al., 2006; Chapman et al., 2009). Assessment of splenocytes and circulating peripheral immune cells revealed increased secretion of pro-inflammatory cytokines in experimental models. Post-stroke assessments in humans have shown similar increases in peripheral pro-inflammatory cytokine (Ferrarese et al., 1999; Landreneau et al., 2018). Despite the initial systemic inflammatory response, the immune response shifts towards immunosuppression, termed stroke-induced immunosuppression (SIIS). As emerging key contributors to SIIS, immunosuppressive neutrophils and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are proposed to induce systemic immunosuppression just days after stroke (Xie et al., 2023). Increases in PMN-MDSCs are reported as early as 24 h after experimental middle cerebral artery occlusion in mice (Kawano et al., 2019). Given that these cells suppress T cell activation and proliferation in cancer, they may play similar roles in immunosuppression after stroke.

Previous studies have reported sex differences in stroke pathology, especially relating to the immune response to stroke. Here we will highlight a few examples; however, we acknowledge this topic has previously been reviewed in depth by others (Ahnstedt and McCullough, 2019; Banerjee and McCullough, 2022; Liu et al., 2022; Tariq et al., 2023). McCullough and others found sex differences in pathways leading to cell death after middle cerebral artery occlusion (MCAO) in mice (McCullough et al., 2005). Specifically, cell death as a result of poly-ADP ribose polymerase (PARP) activation and nitric oxide toxicity is mainly restricted to males, whereas cell death for females is caspase-mediated. Another group revealed that males exhibit increased macrophages and T cells within the ischemic hemisphere compared to females 2 days after MCAO (Xiong et al., 2015). Furthermore, gene expression analysis of whole blood samples from human patients revealed females express significantly more neutrophil specific transcripts up to 3 h following cardioembolic stroke (Stamova et al., 2014). Additional research is needed to elucidate the underlying cellular mechanisms that lead to sex differences in the pathological responses to stroke, including sex differences in cells of the BBB and infiltrating immune cells. Future studies should also aim to determine the extent of hormonal and chromosomal influences that impact sex specific immune responses to stroke.

Whole blood is composed of both cellular (white blood cells, red blood cells) and acellular (plasma, platelets) components. Evaluation of serum biochemistry (i.e., the level of various proteins, enzymes, and lipids) or complete blood count (CBC, i.e., cellular counts and proportions) can yield insight into potential toxicity of therapeutic systems. Several recent reports suggest that biochemical and CBC responses to nanoparticle administration depend on sex (Gosselin, Ramaiah, and Earl, 2011). Lanthanum titanate nanoparticles (LT NPs) produced differing effects on both serum markers and CBC as a function of biological sex, with evidence for immunosuppression in male mice under the same conditions in which expansion of monocytes was observed in female mice (Akram et al., 2020). Male mice were also observed to respond to nanoparticle administration with increased triglyceride levels, which was not observed in females and that may raise concerns for patients at-risk for cardiovascular disease. Sex differences were observed for female versus male rats after treatment with CuNPs, particularly at high doses, and these differences were not observed in the free ion Cu (non-nanoparticle) groups (Riaz et al., 2020). Complex and sex-dependent effects on serum biochemistry for female versus male rats following administration of CuNPs have been reported by other investigators (Kim et al., 2016). Chen et al. described differences in thymus and spleen indexes for female and male mice following administration of PEGylated AuNPs, suggesting sex-dependent immunological response (Zhang et al., 2013). These differences included distinct immunological responses, as well as differences in white blood cell, red blood cell, and platelet counts that depended on dose as well as sex. In other work, male mice that received oral administration of ZnO nanoparticles showed no change in serum biochemistry, while females showed increased bilirubin compared to their control, which could suggest sex-dependent liver toxicity (Kuang et al., 2021). Sex-dependent physiological reactions to nanoparticle treatment can involve direct impact on the endocrine system; when female rats were dosed with titanium dioxide nanoparticles, subjects exhibited transient hypoglycemia on multiple days of observation, while the same dosing regimen in male rats did not cause hypoglycemia (Chen et al., 2020). Sex differences in insulin and glucagon levels likely explain these results and could be a critical sex-dependent safety consideration for individuals with aberrant glucose metabolism (e.g., in diabetes). These data support an expectation that serum biochemistry and CBC responses to nanomedicine will depend on biological sex, which is an important consideration in toxicological evaluation of nanoparticle systems.

Oxidative stress is a major focus of CNS research in acute brain injury, being both a driver and potential therapeutic target for disease pathology (Allen, C. L. and Bayraktutan, U., 2009; Rodriguez-Rodriguez et al., 2014; Salim, 2017; Hakiminia et al., 2022). Oxidative stress involves an imbalance between reactive oxygen species (ROS) and antioxidant cellular machinery, leading to an accumulation of ROS that yield damage to intracellular lipids, proteins, and oligonucleotides. Increased oxidative stress has been linked causally to chronic inflammation and neurodegenerative processes in Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS) (Pizzino et al., 2017). This mechanism of cellular and tissue damage is increasingly implicated in the secondary injury cascades that result from acute CNS, which is discussed in greater detail in Section 5.1.2 of this review. Early evidence suggests that oxidative responses to nanoparticle administration depend on sex, although the reported relationships are complex and are not at a stage where they can be generalized. In one study, researchers studied biocompatibility of low- and high-dose (LD and HD, respectively) PVP-coated AgNPs (Ćurlin et al., 2021). Females and males displayed distinct patterns of peroxyl radical accumulation that depended both on the particle type as well as the organ system examined. Interestingly, female liver and kidneys both showed a decrease in superoxide dismutase (SOD) levels for the LD group and a return to control values in the HD group. In contrast, levels of SOD in male rats did not show a clear pattern between liver and kidney or between HD and LD. In separate work utilizing a nanoparticle with specific intent of reducing catalase and SOD, significant reduction of oxidative stress in male mice was achieved with lower ratios of SOD1:CAT in nanoparticles, whereas the ratio was doubled in females to achieve the same effect (Tarudji et al., 2023). Complex antioxidant responses were observed to also depend on sex in specific organ systems, including heart, liver, and kidney for lanthium titanate NPs (Akram et al., 2020). Treatment with AgNPs nanoparticles yielded significant changes in oxidative stress markers, such as peroxyl radicals and superoxide radicals, in male and female mouse kidneys, livers, brains and lungs. Differences were not only observed between male and female mice, but there were also differences based on whether the mice were intact or gonadectomized, which highlights the important role that gonadotropin signaling may play (Tariba Lovaković et al., 2021). Taken in sum, these data suggest that oxidative stress responses can be sex-specific, and that these differences should be considered for the development of new therapeutics.

Growing evidence suggests that sex differences play a role in driving phenotypic, organ-level, or cellular toxicity. In one recent and high profile example, major differences have been observed for tolerability of the solid lipid nanoparticle COVID-19 vaccine, with females reporting significantly higher adverse effects immediately following inoculation and males experience higher susceptibility to adverse cardiac events at later time points (Duijster et al., 2023); the fact that the vaccine is reported to induce cycle irregularities in menstruating individuals (Rahimi Mansour et al., 2023) suggests that endocrine or gonadotropic signaling may be involved. We note that, given the recency of these observations, sex differences in COVID-19 vaccine tolerability is an area of active clinical investigation that is evolving rapidly. Preclinically, sex differences in toxicity have been studied in various formats. Sex differences were observed for inhaled Titanium Dioxide nanoparticles (TiO₂-NPs), such that male rats exposed to TiO₂-NPs exhibited a significant increase in circulating neutrophils, while female rats were apparently more susceptible to toxicity at the organ level (Yamano et al., 2022). Phenotypic evidence of toxicity was evident as pulmonary dust foci (PDF), which presented as milky white spots on the lungs and were proposed to be agglomerations of macrophages; PDFs were seen in females at lower NP doses compared to males, which suggests that females may be more sensitive to the toxicities related to TiO₂ inhalation. Similarly, female rats were observed to be more susceptible to hepatic toxicity, with increased levels of biomarkers for oxidative stress after oral administration. In contrast to these results, a different study observed that females were less susceptible to lung toxicity than males following exposure to ZnO-NPs (Sehsah et al., 2022); although the initial experiments would have suggested a simple or generalizable dependence of toxicity on sex, their deeper analyses that examined genes regulating oxidative stress responses and inflammation-associated chemokines revealed highly complex regulation of individual pathways. Yang et al. demonstrated that there were significant changes in hormone production in the female rats treated with copper nanoparticles (CuNPs), as well as heightened expression of caspase proteases following parenteral administration in females, although they did not compare these results to male mice (Yang et al., 2017). Other research has also shown an increase in micronucleation of immune cells, with a higher quantity of these cells in male versus female rats across multiple dosing groups by the oral route (Kim, Soon Yong et al., 2008). In detailed studies performed by Han and others, amorphous silica nanoparticles (ASiNPs) instilled intratracheally yielded the highest level of lung damage in female rats compared to males (Han et al., 2020). Expression of caveolin-1 and matrix metalloproteases in females were suggested to account for these differences. In separate studies, cytokine marker expression levels depended on sex. Gokulan and others reported that a T-cell marker was significantly increased in male tissues after 24 h, but the same marker was significantly decreased in females at the same time point and dose (Gokulan et al., 2020). Taken in sum, these data support an expectation that toxicity of nanoparticle systems will depend on sex, although clearly these relationships are complex and may be unique to each nanoparticle systems.