Biology of Sex Differences

In mammals, sex differences in the brain arise from genetic and hormonal factors, including organizational effects of perinatal testosterone. Epigenetic mechanisms including DNA methylation and demethylation have emerged as critical mediators of brain masculinization; specifically, their regulatory enzymes are upregulated in neonatal mice during the critical period of sexual differentiation, with their inhibition abolishing sex-specific cellular phenotypes. Here, we assessed sex differences in gene expression of the DNA demethylation machinery (Tet1, Tet2, Tet3, Gadd45a, Gadd45b and Tdg) during and after the critical period, and examined how these differences relate to the oxytocinergic system. mRNA expression was measured in the prefrontal cortex (PFC), preoptic area (POA) and paraventricular nucleus of the hypothalamus (PVN) at postnatal day (P) 7 and P18. At P7, males showed higher expression of all six genes than females in PFC, with no differences in POA or PVN; by P18, no regional differences remained. Oxytocin (OXT) immunoreactivity was surveyed across periventricular nucleus (Pe), anteroventral periventricular nucleus (AVPe), POA, PVN and supraoptic nucleus (SON). OXT was undetectable in the POA, AVPe and Pe at P7, and no sex differences were found in PVN or SON at either age, or in AVPe at P18. At P18, females showed higher OXT-immunoreactivity in the Pe and POA, than males. For Oxtr, qPCR revealed higher mRNA expression in the PFC of males at P7, with no other regional differences and none remaining at P18. Together, these findings suggest that sex differences in oxytocinergic regions arise from sex-specific epigenetic regulation during the critical period, and that perinatal testosterone may program DNA methylation dynamics underlying sex-specific gene expression in the developing brain. Our results support a model in which testosterone-dependent epigenetic mechanisms contribute to the sexual differentiation of neuroendocrine circuits, linking hormonal signals to long-term brain organization.

Osteoarthritis (OA) disproportionately affects women, and estrogen has been implicated in cartilage and joint homeostasis, yet its effects on meniscal fibrochondrocytes (MFCs), and whether those effects differ by sex, remain poorly defined. We reanalyzed a publicly available RNA-sequencing dataset (Gene Expression Omnibus, GSE199087) comprising human MFCs from a male and a female donor treated with 17{beta}-estradiol (E2) or vehicle. Differential expression, Gene Set Enrichment Analysis, Enrichr, and iLINCS were integrated to identify the transcriptional programs modulated by E2 in each sex. In female MFCs, E2 upregulated pathways governing DNA replication, cell-cycle progression, and genomic maintenance (e.g., MCM8, BRCA1, RAD51), while downregulating inflammatory and extracellular matrix-degrading genes, including MMP1, IL12A, CXCL12, and MYD88. In male MFCs, E2 instead upregulated chromatin remodeling and developmental signaling programs, led by SRCAP, ERCC6, and NOTCH1, and downregulated antigen presentation and mitochondrial genes. These divergent responses indicate that E2 engages distinct, sex-specific transcriptional programs in MFCs, with the female profile favoring proliferation and matrix preservation. Although derived from a limited sample and therefore hypothesis-generating, these findings nominate candidate mechanisms underlying sex differences in meniscal biology and OA susceptibility, and warrant validation in larger, sex-balanced cohorts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/740612v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16026fforg.highwire.dtl.DTLVardef@1a048acorg.highwire.dtl.DTLVardef@3246corg.highwire.dtl.DTLVardef@4c598b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by deficits in social communication and interaction, and restricted interests or repetitive behaviors. ASD is approximately four times more prevalent in males than in females. In this study, we investigated whether sex hormones or sex chromosomes underlie the male bias in ASD susceptibility. Methods: We used the MG4E mouse model, in which microglial eIF4E overexpression produces robust male-biased ASD-like phenotypes. To distinguish the contributions of sex hormones and sex chromosomes, MG4E mice were crossed with four-core-genotype (FCG) mice carrying Sry gene manipulations, generating eight genotypes of experimental mice. Social interaction and repetitive behaviors were assessed using standard behavioral assays. Dendritic spine density was quantified in Thy1-GFP mice. Expression of estrogen receptors (ERs) and androgen receptor (AR) was examined in microglia isolated from control and MG4E mice. Results: Sex hormones, rather than non-Sry genes on sex chromosomes, are responsible for the male-biased deficits in social interaction and the increase in dendritic spine density. At postnatal day 14, ERs were undetectable in microglia, whereas ER expression was readily detected in neurons. Conclusions: These findings demonstrate that sex hormones are a major determinant of the increased susceptibility of males to ASD-like phenotypes in MG4E mice. The absence of ER expression in microglia suggests that sex hormones may act indirectly through hormone-responsive neurons to regulate microglia-neuron interactions during neurodevelopment.

AimThe aim of the present study was to determine whether pharmacological inhibition of aldosterone synthesis during the stress-hyporesponsive period (SHRP) affects behaviour and adrenocortical stress responsiveness later in development and whether these effects differ between males and females. MethodsNewborn Wistar rat pups (males n=40, females n=40) were treated with aldosterone synthase inhibitor FAD286 (30 mg/kg per day, orally) or vehicle from PND3 to PND9. To verify the pharmacodynamic action of FAD286, serum and adrenal glands from 10-day-old pups were analysed. The remaining pups were weaned on PND21 and underwent open-field (PND23), elevated plus-maze (PND29) and salt-preference testing. At PND46, half of each group was exposed to restraint stress for 120 min. ResultsIn 10-day-old pups, treatment with FAD286 resulted in increased gene expression of CYP11B2 (aldosterone synthase) and CYP11B1 (11-beta-hydroxylase) in the adrenal glands, increased serum levels of corticosterone, and decreased concentrations of serum aldosterone. FAD286 did not modify the general locomotor activity assessed in juvenile rats. Inhibition of aldosterone synthase by FAD286 resulted in altered anxiety-like behaviour in a sex-dependent manner. Postnatal FAD286 treatment led to increased anxiety-like behaviour in female, but not male rats. During adolescence, early FAD286 treatment increased overall aldosterone concentrations without altering the aldosterone response to restraint. Basal corticosterone concentrations were unchanged, whereas the response to restraint was enhanced. ConclusionsThe present study demonstrates that transient inhibition of aldosterone synthesis during the SHRP led to alterations in anxiety-related behaviour and adrenocortical regulation later in development, with some behavioural effects being sex-dependent.

Sex-specific differences in COVID-19 outcomes are well documented, with male sex being a key risk factor for severe disease. Multiple factors, including differences in the expression of Interferons (IFNs) and Interferon stimulated genes (ISGs) between the sexes have been identified as contributing to these differences. Adenosine-to-inosine (A-to-I) RNA editing, mediated by ADAR enzymes, is an important component of antiviral innate immune signaling. While previous studies, including us, have identified altered expression and activity of ADARs in response to SARS-CoV-2 infection, it remains unknown whether sex-specific differences in expression and activity of ADARs exist during infection with SARS-CoV-2. We address this question by analyzing a publicly available whole blood RNA sequencing dataset from males and females, matched for age and comorbidity status across pre-, mid-, and post-infection stages of SARS-CoV-2 infection. Our results show distinct patterns of ADAR expression between the sexes across different stages of infection, with significant differences in the expression of ADAR and ADAR2 in pre- and mid- infection that were not observed post-viral clearance. Interestingly, expression of predominantly neural isoform ADAR3 was significantly different and higher in females, mid-infection. Furthermore, these differences in ADAR expression were likely amplified downstream with significant differences in the global editing levels. Correlation analysis revealed that the relationship between ADAR expression and global editing activity differed by sex and stage of infection, indicating that ADAR editing is dynamically regulated and is influenced by factors other than ADAR transcript abundance. Noteworthy, sex-specific differences in ADAR editing were observed within genes that escape X-chromosome inactivation (XCI), a gene set enriched in immune related functions and known to be involved in sex-specific outcomes to viral infections. ADAR editing within these genes differed at baseline and changed across stages of infection in a manner independent of gene expression. Altogether, our findings establish ADAR editing as an additional layer of immune regulation that may be contributing, in part, to the differential clinical manifestations of COVID-19 observed between males and females.

Chronic alcohol consumption is associated with increased breast cancer risk; however, the long-term effects of alcohol exposure on DNA damage response (DDR) signaling and therapeutic vulnerability in breast epithelial cells remain incompletely understood. In the present study, we established a chronic alcohol exposure model using immortalized human breast epithelial MCF-10A cells to investigate persistent alterations in DNA damage signaling and sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition. MCF-10A cells that chronically exposed to 0.2% alcohol for 20 weeks and then withdrew alcohol treatment for 4 weeks still retained elevated {gamma}H2AX levels and sustained activation of ATM/ATR-associated DDR pathways. Chronic alcohol-exposed cells also exhibited increased sensitivity to the PARP inhibitors olaparib and veliparib, as demonstrated by reduced cell viability, decreased clonogenic survival, and enhanced apoptosis. Mechanistically, olaparib induced greater G2/M checkpoint activation and replication-associated DNA damage in chronic alcohol-exposed cells compared with control cells. In addition, chronic alcohol exposure increased PARP expression and enhanced activation of DDR signaling following PARP inhibition. These findings demonstrate that chronic alcohol exposure induces persistent DNA repair stress and sensitizes breast epithelial cells to PARP inhibition. Our study suggests that chronic alcohol-associated genomic stress may create potential vulnerability to PARP inhibition.

Background: Prenatal cannabis use is increasing, yet the effects of inhaled cannabis smoke on placental molecular function remain poorly defined. Objective: To characterize placental transcriptomic alterations following prenatal cannabis smoke exposure using a physiologically relevant murine inhalation model. Methods: Timed-pregnant CD-1 mice were exposed to cannabis cigarette smoke or room air from embryonic day (E)6.5 to 18.5. Placentae were collected at E18.5 for microarray-based transcriptomic profiling. Differential gene expression was assessed using fetal sex adjusted linear models and false discovery rate correction. Gene set variation and ranked pathway enrichment were performed to evaluate compartment-associated and biological transcriptional programs. Selected genes were validated by quantitative PCR. Results: Large-scale transcriptomic profiles revealed no clear visual clustering by exposure; however, 23 genes were differentially expressed (FDR <0.05, |log2 fold change| > 0.5). Labyrinth-associated transcriptional signatures were reduced, whereas junctional-associated signatures were increased in exposed placentae. Pathway analyses revealed negative enrichment of fatty acid metabolism, bioenergetic, and transmembrane transport pathways, alongside positive enrichment of RNA processing and transcriptional-regulatory pathways. Quantitative PCR confirmed altered expression of multiple transport-related genes. Electron transport chain-associated gene expression was altered despite preserved placental respiratory measurements. Conclusions: Prenatal cannabis smoke exposure is associated with subtle yet coordinated placental transcriptional alterations involving metabolic, transport-associated, and gene-regulatory pathways, alongside compartment-associated transcriptional shifts. These findings provide insight into placental responses to whole cannabis smoke exposure and identify pathways that may contribute to altered placental function during pregnancy.

The human bony labyrinth has attracted increasing interest because of its taxonomic, evolutionary, and functional significance. Although sexual dimorphism has been reported in several aspects of the temporal bone, the extent to which sex, age, size, and allometry contribute to labyrinth shape variation remains poorly understood. Here, we investigated patterns of sexual shape variation in the human bony labyrinth using three-dimensional geometric morphometrics in a sample of 98 archaeological individuals from South America with known genomic sex. Centroid size and allometric effects were assessed in a subset of 90 individuals with comparable metric scaling. In addition to analysing the complete labyrinth, the cochlea and semicircular canals were examined separately to evaluate region-specific patterns of sexual shape variation. Principal Component Analysis showed extensive overlap between females and males, and overall labyrinth shape did not differ significantly between sexes. Males exhibited significantly larger labyrinths than females, and centroid size explained a small but significant proportion of overall shape variation. Regional analyses showed no evidence of significant sexual shape differences in the cochlea or in any individual semicircular canal when analysed separately. In contrast, the combined semicircular canal system exhibited subtle but significant sexual shape variation independent of centroid size, whereas morphological disparity did not differ between sexes. The geometric comparison of the female and male consensus configurations further showed that sexual shape variation was regionally heterogeneous. Whereas the cochlea exhibited a pattern of localized changes with low directional coherence, the semicircular canals displayed more coordinated regional shape changes. The male consensus also exhibited slightly higher canal circularity across all three semicircular canals, particularly the posterior canal, while differences in canal-plane orientation remained minimal. These findings demonstrate that sexual shape variation in the human bony labyrinth is subtle and anatomically partitioned among its components. Although significant sex differences in centroid size were detected across most anatomical regions, overall labyrinth shape and cochlear morphology were primarily influenced by allometry, whereas significant sex-related shape differences were detected only when the semicircular canals were considered as an integrated anatomical system. These findings demonstrate that sexual dimorphism in the human bony labyrinth is subtle but regionally heterogeneous, with the cochlea and semicircular canals exhibiting distinct patterns of shape variation, suggesting that these structures are influenced by different developmental, functional, and evolutionary processes.

Background: Thyroid disorders represent a substantial endocrine disease burden across South Asia, yet systematic epidemiological data from specific regions of Bangladesh remain scarce. The objective of this study was to determine the prevalence and demographic distribution of thyroid stimulating hormone (TSH) abnormalities among hospital outpatients in Jashore district, southeastern Bangladesh. Methods: A cross-sectional survey was conducted among 200 consecutive outpatients presenting for thyroid evaluation at LabAid Hospital, Jashore, from October 2017 to December 2017. Serum TSH concentrations were measured, and participants were classified as euthyroid, hypothyroid, or hyperthyroid based on standard reference intervals, then stratified by sex and seven age categories. Results: Of the 200 participants (170 female, 30 male), 101 (50.5%) showed abnormal TSH values. Hypothyroidism was identified in 97 participants (48.5%) and hyperthyroidism in 4 (2.0%). The overall prevalence of thyroid dysfunction was nearly identical between sexes (female 50.6% vs male 50.0%, P=1.00), while age group showed a highly significant association with TSH abnormality (P<0.001), peaking in the 40-49-year group (89.7%). The female-to-male ratio for total thyroid dysfunction was 5.7:1, reflecting the much larger proportion of female participants rather than a higher within-sex risk. Conclusion: Thyroid dysfunction, principally hypothyroidism, is highly prevalent among hospital outpatients in Jashore City, with prevalence rising sharply with age and peaking in middle adulthood, supporting the case for systematic age-targeted thyroid screening in this population.

Early-life adversity during critical developmental windows markedly increases vulnerability to neuropsychiatric disorders later in life. Although adult behavioral and neurobiological alterations following early stress are well documented, the early emerging phenotypes and their underlying cellular and molecular mechanisms during juvenile development remain poorly understood. In this study, we utilized the translational Scarcity-Adversity Model (SAM) to investigate early behavioral alterations and hippocampal neuroplasticity in juvenile offspring. We characterized the behavioral profile of juvenile subjects exposed to SAM to identify early manifestations of vulnerability. Next, we evaluated hippocampal neurogenesis by quantifying neural progenitor cell proliferation immediately following stress exposure (at postnatal day 13, PND 13) and the short-term survival of newborn neurons (1 week post-labeling) in the dentate gyrus of juvenile offspring. Furthermore, given the functional compartmentalization of the hippocampus, we measured the protein expression of mature BDNF (mBDNF) and its precursor proBDNF across both dorsal and ventral domains. At the behavioral level, SAM-exposed juvenile offspring exhibited increased immobility in the forced swim test alongside impaired spatial memory in the novel object location task. Regarding hippocampal neurogenesis, SAM exposure did not alter immediate progenitor proliferation at PND 13; however, it produced a significant, male-specific reduction in newborn neuron survival, concomitant with a trend toward decreased mBDNF levels in males. Together, these results provide critical insight into the early neurodevelopmental trajectories triggered by early-life adversity, identifying early behavioral impairments and male-specific neuroplastic deficits as key juvenile markers of vulnerability preceding adult psychopathology.

The success of modern antiretroviral therapy (ART) has increased the life expectancy of people living with HIV to levels approaching that of uninfected individuals. For women living with HIV (WLWH), this means that more will survive to undergo menopause and experience the consequences of decreased ovarian hormone levels, particularly estrogen (E2). The recent change in federal guidance for use of postmenopausal hormone therapy is increasing demand for both E2-alone and E2+progestogen formulations to control adverse symptoms of menopause. The consequences and efficacy of hormone therapy in WLWH are thus an important issue for WLWH and their healthcare providers. The role of E2 replacement in postmenopausal WLWH is a significant issue because of its potential effects on control the viral reservoir and its demonstrated beneficial metabolic effects in uninfected postmenopausal women. To address these questions, we employed a novel nonhuman primate model of postmenopausal WLWH undergoing E2 replacement. Reproductively competent female rhesus macaques were infected with simian immunodeficiency virus (SIV) and then subjected to a daily ART regimen. After complete suppression of plasma viremia, all animals were ovariectomized (OVX) and then implanted with Silastic capsules containing either cholesterol vehicle or sufficient E2 to restore pre-OVX plasma levels. Plasma and cell-associated viral dynamics, immune responses, body composition, systemic and tissue-specific metabolic parameters, cytokine profiles, and parameters of bone health were followed longitudinally from baseline through 34 weeks of E2 deficiency or replacement. We found that E2 status did not significantly affect plasma or tissue viral dynamics or overall metabolic homeostasis. However, E2 replacement exerted beneficial effects on several aspects of bone health in spite of a chronic inflammatory state that persisted following effective ART suppression of the SIV reservoir. Our findings suggest that hormone therapy, specifically E2 replacement, offers benefit to WLWH, particularly with respect to bone loss.

Preeclampsia (PE) is a severe pregnancy-associated hypertensive disorder and a major contributor to maternal and perinatal morbidity and mortality. The mechanisms of PE pathogenesis are not yet understood. This paper aims to explore candidate PE-associated biomarkers and regulatory mechanisms using bioinformatics analysis of placental transcriptomic datasets. We downloaded placental transcriptomic datasetsGSE203507 and GSE148241 from GEO and investigated differentially expressed genes(DEGs) between PE and control samples. We performed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO), and protein-protein interaction (PPI) analyses to characterize the potential role, disease associations, and interaction networks of the DEGs. We validated the mRNA expression patterns of candidate hub genes using two independent placental transcriptomic datasets, GSE143966 and GSE114691.We identified 263 DEGs in the discovery analysis and further obtained 150 overlapping DEGs including80 upregulated genes and 70 downregulated genes, for downstream analysis. We then identified eight candidate hub genes: OPRK1, OXGR1, HCAR3, CCR5, HCAR2, CXCL1, FPR3, and SSTR1using Meta scape software(v3.5.20260201). In the validation phase, most candidate hub genes showed broadly consistent mRNA expression trends across GSE114691 and GSE143966, while FPR3 showed weaker cross-dataset consistency. These findings provide candidate PE-associated genes and regulatory pathways for further experimental and clinical validation..

Single cell differential expression analysis enables biologists to make statistical conclusions about which genes are up or downregulated in a particular cell type, between two conditions, such as those with or without a disease. However, due to biological and technical noise, these differences are hard to detect reliably. Determining if an experiment has sufficient power is often derived from simulations or small pilot samples, if done at all. Here, we use sex-biased differential expression on 1,494 donors in three brain cell types to derive empirically grounded power estimates for a variety of experimental setups. Our work reveals a substantial lack of power for reliably detecting the small effects in the range that many studies report, even in experiments containing 600 donors. When reducing the astrocyte data to the poor sequencing characteristics of microglia, over half the differentially expressed genes (DEGs) detected in the full set were lost, highlighting the damage caused by insufficient sequencing depth and cell counts. Despite standard thresholds of adjusted p-value with multiple testing correction, only the top quartile of significant genes were reproducible. Furthermore, from fitting a predictive model to the empirical outcomes, we find cell count and expression levels of genes to be a strong determinant of power. As such, we advocate for future studies to employ cell type enrichment and deeper sequencing, especially for rarer populations like microglia, emphasise the importance of powering experiments of this type when seeking robust findings, and suggest stricter significance thresholds for future discoveries.

Prenatal stress influences brain development; however, its long-term, region-specific effects on brain aging in males and females remain insufficiently characterized. This study examined the impact of unpredictable mild prenatal stress on behavior, cognition, and maternal care in C57BL/6J mice at middle (12 months) and old (18 months) ages. Stressed dams showed fragmented, unpredictable maternal behaviors, creating a stressful postnatal environment without impairing caregiving capacity. Both male and female offspring were born with lower birth weights and showed rapid postnatal growth, a trajectory associated with central insulin resistance and hippocampal signaling deficits. Sex-specific behavioral differences emerged with age. Middle-aged stressed males exhibited anhedonia, which progressed to hyperactivity, stereotypy, and persistent spatial memory deficits in old age. These outcomes corresponded with accelerated fronto-striatal decline, likely mediated by insulin resistance and increased rigidity of medial prefrontal cortex structures due to diminished local estrogenic activity. In contrast, middle-aged stressed females displayed hypoactivity and reduced anxiety, later developing anhedonia in old age while maintaining hippocampus-dependent memory. In females, this pattern supports a model in which a neuroestrogen - and growth factor-dependent feedback loop transiently preserves hippocampal plasticity but compromises dopaminergic function in reward circuits. This balance may deteriorate with reproductive senescence. Both sexes retained cognitive flexibility, indicating domain-specific rather than global cognitive impairment. These findings demonstrate that excessive prenatal glucocorticoid exposure and unpredictable postnatal care produce a combined hormonal and metabolic insult, resulting in sexually divergent trajectories of brain aging. The results underscore the necessity of incorporating sex as a biological variable in fetal programming research across the lifespan.

INTRODUCTIONSex differences in mental health emerge during adolescence, a period marked by the onset and the establishment of menstrual cycle. However, is rarely examined how menstrual cycle regularity, a marker of hormonal function, modulates mental health. OBJECTIVEto analyse sex differences in mental health symptoms among adolescents considering the menstrual cycle regularity and sleep. METHODSA three-group design (female students with regular cycles/FR, n=77; with irregular cycles/FI, n=59; and male students/M, n=76) in a sample of Brazilian high-school adolescents (n=212; 14-18 years) enrolled in morning and full-time classes was used to test the hypothesis that mental health symptoms follow a graded pattern across these groups. RESULTSMean DASS-21 scores across all groups fell at or above the Mild severity threshold for mental health subscales. GLMs confirmed a monotonic gradient increase in group order (M[-&gt;]FR[-&gt;]FI) which was associated with higher scores on all outcomes (stress {beta}/step=4.33, p<.001; anxiety {beta}/step=4.08, p<.001; and depression {beta}/step=2.34, p=.010; model R{superscript 2}=.16, .13, .08 respectively). However, no differences were observed in sleep duration, social jetlag, chronotype, sleep quality, or sleep-debt. Then, a secondary analysis assessed sex-specific associations between socioeconomic status (SES) and mental health; higher SES was inversely related to stress, anxiety, and depression, being protective only in males (stress Males {beta}=-2.68, p=.011/Females {beta}=0.46, p=.614). CONCLUSIONThese findings support the reframing of menstrual irregularity not only as a reproductive health concern but also as a biological determinant of mental health risk in female adolescents, a vulnerability that sleep disruption and socioeconomic resources do not adequately explain.

Recurrent urinary tract infection represents a substantial unmet public health in women. Local administration of estradiol has been shown to reduce recurrence, however in vitro models of the female urinary tract remain limited and the mechanisms underlying the effects of estradiol are incompletely understood. Here, we describe a novel iPSC organoid differentiation protocol and its application to establish a multilayered transwell barrier culture model. Estradiol treatment resulted in reduced expression of innate antimicrobial peptides and cytokines, together with increased expression of demannosylation pathways. Treatment of transwell cultures with a combination of female sex hormones reduced endogenous CXCL8 signaling, independently of a 24-hour uropathogenic Escherichia coli (UPEC) challenge. To our knowledge, this is the first iPSC organoid-derived model of the urinary tract, which provides a platform for investigating interactions between the urothelium, urobiome and hormonal environment.

Features of urbanicity have been linked to poor psychiatric health in adulthood, but little is known about their relationship with biological age and cognition earlier in life. Using the Avon Longitudinal Study of Parents and Children (ALSPAC), we tested associations between the built environment and epigenetic ages (EpiAgeAltum, EpiAgeHorvath, EpiAgePACE) (n=2,452), brain age (n=751), and cognition (n=12,199). Analyses were replicated in Generation R and triangulated in the Millennium Cohort Study. The built environment was not associated with epigenetic ages ({beta}Altum range = 0.013-0.015, p range = .466-.689; {beta}Horvath range = -0.011-0.023, p range = .182-.728; {beta}PACE range = -0.045-0.013, p range = .131-.524) or brain age ({beta} range = 0.022-0.028, p range = .337-.429). A negative association with cognition was observed in Generation R. We provide no consistent evidence that childhood built environment exposure relates to biological age and cognition in adolescence, suggesting these relationships may emerge in later life.

Background: Across phenotypic and genotypic studies, executive functioning (EF) has been shown to be associated with externalizing behaviors, including attention-deficit/hyperactivity disorder (ADHD), aggression, and substance use, and is a proposed underlying mechanism of these behaviors. However, there is limited research on the shared biological mechanisms underpinning EF and externalizing behaviors, including their overlap at the level of genomic regions. Methods: The current study used genome-wide association study summary statistics of EF, ADHD, aggressive behavior, alcohol use, and alcohol use disorder to examine whether the heritability of these traits or the co-heritability between them is enriched for genes expressed in particular tissues or stratified by other annotation types. Additionally, the study examined the genomic regions contributing to the covariation between EF and the externalizing traits to identify genetic variants and genes that may be underlying their overlap. Results: First, across all phenotypes, there was evidence for enrichment of genetic signals in DNase hypersensitive sites and H3K4me1 regions, and significant enrichment among genes expressed in the anterior cingulate cortex and frontal cortex. Second, when combining results across regional methods, there was evidence for associations between EF and each of the externalizing behaviors in regions on chromosomes 1, 11, and 15. Conclusions: These findings highlight biological pathways that may link the relations between EF and externalizing behaviors and provides additional evidence of EF as an underlying mechanism of externalizing behavior.

Ketogenic (keto) diets have gained popularity due to their potential benefits on weight loss and metabolic conditions. However, the consequences of consuming a keto diet before, during, and after pregnancy on maternal and offspring health remain incompletely understood. To investigate this, female Swiss Webster mice were randomly assigned to either a keto or control diet. After 6 weeks on diet, females were mated and maintained on their respective diets throughout pregnancy and lactation. In experiment 1, glucose tolerance tests (GTT) were performed on pregnant dams at gestational day (GD) 16.5. At GD 17.5, dams were euthanized, urine was collected for ketone analysis, and fetal and placental weights were recorded. In experiment 2, dams delivered naturally and one male and one female offspring per dam were retained and weighed weekly. At 12 weeks of age, offspring underwent GTT, followed by euthanasia and serum collection for insulin and leptin analysis. Compared with controls, keto dams exhibited increased body weight, impaired glucose tolerance, elevated urinary ketones, and reduced circulating insulin during late gestation (p<0.05). Fetuses from keto dams were significantly smaller (p<0.05), whereas placental weight and placental efficiency were unchanged. Offspring from keto dams weighed less than controls during early postnatal development but demonstrated catch-up growth by 12 weeks of age. At 12 weeks, keto offspring exhibited a modest but statistically significant improvement in glucose tolerance compared to control offspring (p<0.05). Male keto offspring had significantly decreased serum insulin (p<0.05) compared to male control offspring, while no differences were observed in females. Serum leptin levels did not differ between diet groups, although sex differences present in control offspring were not observed in keto offspring. Together, these findings suggest that maternal ketogenic diet consumption is associated with altered maternal glucose homeostasis during pregnancy and reduced fetal growth in this mouse model. Although offspring exhibited only modest metabolic alterations during early adulthood, the observed changes in growth trajectory and sex-specific metabolic profiles support further investigation into the long-term consequences of maternal ketogenic diet exposure during pregnancy.

Background: Helsmoortel-Van der Aa syndrome, caused by pathogenic variants in ADNP, is characterised by substantial clinical heterogeneity, but how biological sex influences disease biology and treatment response remains an emerging area of investigation. Here, we investigated whether sex modifies the behavioural and molecular consequences of Adnp deficiency, the response to the investigational drug candidate davunetide (NAP), and the clinical phenotype of Helsmoortel-Van der Aa syndrome. Methods: We studied male and female Adnp heterozygous mice harbouring the p.Leu822Hisfs*6 variant and sex-matched wild-type littermates using continuous 24-h behavioural phenotyping, hippocampal genome-wide DNA methylation and bulk RNA sequencing. NAP effects on behaviour, hippocampal ADNP protein abundance, and epi-transcriptomic responses were assessed using genotype-by-treatment models. Four Core Genotypes mice were assessed to distinguish sex-chromosome and gonadal contributions to hippocampal Adnp transcript expression, and reproductive-tissue transcriptomes were analysed in Adnp mice to unravel a possible endocrine component of the disease. Findings were compared with clinical and developmental data from 129 individuals with Helsmoortel-Van der Aa syndrome. Findings: Adnp deficiency produced a shared overall behavioural phenotype in male and female mice, but through distinct patterns of behavioural disruption, indicating that sex modifies the implementation rather than the magnitude of the phenotype. This distinction was also evident at the molecular level: hippocampal DNA methylation was predominantly determined by genotype and showed convergence between sexes, whereas transcriptional responses diverged, with mitochondrial pathways predominating in males and chromatin, RNA processing, and cell-cycle pathways in females. NAP partially shifted behavioural abnormalities towards the wild-type state in both sexes and increased hippocampal ADNP protein abundance in heterozygous mice, without reversing the underlying methylation phenotype. Its transcriptional effects were markedly broader in males, indicating a sex-dependent molecular response to treatment. In reproductive tissues, Adnp deficiency was associated with convergent alterations in steroid hormone biosynthesis, despite no evidence for direct regulation of hippocampal Adnp expression by sex-chromosome complement or gonadal hormonal state. By contrast, sex was not associated with robust differences across the clinical and developmental features assessed in 129 individuals with Helsmoortel-Van der Aa syndrome after correction for multiple testing. Interpretation: Biological sex modifies how Adnp deficiency is expressed at behavioural and molecular levels, but these differences do not necessarily define distinct clinical phenotypes. The convergence of the methylation response alongside greater divergence in transcriptional and behavioural responses identifies sex as a modifier of disease biology rather than a determinant of the core phenotype. The sex-dependent response to NAP further indicates that biological sex may influence pharmacological responses even when treatment-associated behavioural improvement is observed in both sexes. These findings support consideration of biological sex as a prespecified variable in preclinical and clinical studies of Helsmoortel-Van der Aa syndrome and suggest that systematic investigation of sex-dependent biology may also be relevant to therapeutic development across rare diseases. Keywords: Helsmoortel-Van der Aa syndrome (HVDAS), Activity-Dependent Neuroprotective Protein (ADNP), autism spectrum disorder, intellectual disability, sex, mouse model, behaviour.