Precision medicine requires understanding how both sex and gender influence health.

Progress in studying sex as a biological variable (SABV) is slow, and the influence of gendered effects of the social environment on biology is largely unknown. Yet incorporating these concepts into basic science research will enhance our understanding of human health and disease. We provide steps to move this process forward.

A review of the historical use of sex as a biological variable in the American Journal of Physiology-Heart and Circulatory Physiology.

Despite National Institute of Health (NIH) mandates requiring sex as a biological variable (SABV), female underrepresentation persists in research, driving the American Journal of Physiology-Heart and Circulatory Physiology (Am J Physiol-Heart Circ) to publish SABV expectations in 2021. To determine progress within the Am J Physiol-Heart Circ, this mini-review evaluated SABV during the first 6 mo of each decade from 1980 to 2020, and 2019, to mitigate pandemic influence. Of the 1,205 articles published, 1,087 articles were included in this review (articles without original research subjects were excluded), of which 72.9% identified subjects. There were consistently fewer female human participants than males, except within 2019 (1980: females n = 3, males n = 5; 1990: females n = 70, males n = 199; 2000: females n = 305, males n = 355; 2010: females n = 186, males n = 472; 2019: females n = 1,695, males n = 1,550; 2020: females n = 1,157, males n = 1,222) and fewer female animals than males (1980: females n = 58, males n = 1,291; 1990: females n = 447, males n = 2,628; 2000: females n = 590, males n = 3,083; 2010: females n = 663, males n = 4,517; 2019: females n = 338, males n = 1,340; 2020: females n = 1,372, males n = 1,973). Only 16 (12.3%) articles including humans discussed SABV from 1980 to 2020. There are persistent SABV disparities within Am J Physiol-Heart Circ with some improvements in recent years. It is imperative that organizations such as the American Physiological Society and NIH foster an expectation of SABV as the norm, not the exception.

Considering hormones as sex- and gender-related factors in biomedical research: Challenging false dichotomies and embracing complexity.

The inclusion of sex and gender considerations in biomedicine has been increasing in light of calls from research and funding agencies, governmental bodies, and advocacy groups to direct research attention to these issues. Although the inclusion of both female and male participants is often an important element, overreliance on a female-male binary tends to oversimplify the interactions between sex- and gender-related factors and health, and runs a risk of being influenced by cultural stereotypes about sex and gender. When biomedical researchers are examining how hormones associated with gender and sex may influence pathways of interest, it is of crucial importance to approach this work with a critical lens on the rhetoric used, and in ways that acknowledge the complexity of hormone physiology. Here, we document the ways in which discourses around sex, gender and hormones shape our scientific thinking and practice in biomedical research, and review how the existing scientific knowledge about hormones reflects a complex and dynamic reality that is often not reflected outside of specialist niches of hormone biology. Where biomedical scientists take up sex- and gender-associated hormones as a way of addressing sex and gender considerations, it is valuable for us to bring a critical lens to the rhetoric and discourses used, to employ a sex contextualist approach in designing experimentation, and be rigorous and reflexive about the approaches used in analysis and interpretation of data. These strategies will allow us to design experimentation that goes beyond binaries, and grapples more directly with the material intricacies of sex, gender, and hormones.

Deconstructing sex: Strategies for undoing binary thinking in neuroendocrinology and behavior.

The scientific community widely recognizes that "sex" is a complex category composed of multiple physiologies. Yet in practice, basic scientific research often treats "sex" as a single, internally consistent, and often binary variable. This practice occludes important physiological factors and processes, and thus limits the scientific value of our findings. In human-oriented biomedical research, the use of simplistic (and often binary) models of sex ignores the existence of intersex, trans, non-binary, and gender non-conforming people and contributes to a medical paradigm that neglects their needs and interests. More broadly, our collective reliance on these models legitimizes a false paradigm of human biology that undergirds harmful medical practices and anti-trans political movements. Herein, we continue the conversations begun at the SBN 2022 Symposium on Hormones and Trans Health, providing guiding questions to help scientists deconstruct and rethink the use of "sex" across the stages of the scientific method. We offer these as a step toward a scientific paradigm that more accurately recognizes and represents sexed physiologies as multiple, interacting, variable, and unbounded by gendered preconceptions. We hope this paper will serve as a useful resource for scientists who seek a new paradigm for researching and understanding sexed physiologies that improves our science, widens the applicability of our findings, and deters the misuse of our research against marginalized groups.

Novel Correlation between TGF-ß1/-ß3 and Hormone Receptors in the Human Corneal Stroma.

This study investigated the interplay between transforming growth factor beta (TGF-ß1/T1 and TGF-ß3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 106 cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα) and beta (ERß), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERα, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERα, ERß, and GnRHR. T3 caused significant upregulation in expressions PR, ERα, ERß, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERα, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-ß isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms.

Maxillary sinus dimensions in skeletal class II population with different vertical skeletal patterns.

OBJECTIVES: The location of the maxillary sinus significantly affects the orthodontic treatment, particularly when temporary anchorage devices (TADs) are taking place. The current study aims to evaluate the maxillary sinus size and location in a skeletal class II population. MATERIALS AND METHODS: The pre-orthodontic treatment CBCT images of the skeletal class II population were selected. The sinus's volumetric size, height, width, and depth were measured and compared among different skeletal vertical patterns and between genders. In addition, the height and width of the alveolar bone surrounding the maxillary sinus floor were quantified in the same manner. RESULTS: Patients who displayed a high-angle skeletal pattern had significantly greater maxillary sinus dimensions, shorter vertical distance between the maxillary sinus floor and the alveolar bone crest, and thinner alveolar bone surrounding the maxillary sinus. Meanwhile, the maxillary sinus dimension measurements were positively correlated with the SN-MP angle in both genders but only correlated with ANB angle in females. On the other hand, the vertical distance between the maxillary sinus floor and the alveolar bone crest was negatively correlated with the SN-MP angle in males but the ANB angle in females. CONCLUSIONS: In the skeletal class II population, the high-angle patients faced a higher risk of maxillary sinus perforations by TADs. In addition, gender-related variations were noticed warranting clinical attention, as males have a higher potential for maxillary sinus penetration from TAD placement than females. CLINICAL RELEVANCE: Maxillary posterior alveolar TADs are often prescribed to achieve the distalization of maxillary posterior teeth in class II patients. The current study provided more insight into the "safe zone" for TAD placement related to the maxillary sinus.

Animal models built for women's brain health: Progress and potential.

Women and men have different levels of risk for a variety of brain disorders. Despite this well-known epidemiological finding, preclinical work utilizing animal models has historically only included male animals. The policies of funders to require consideration of sex as a biological variable has shifted the momentum to include female animals in preclinical neuroscience and to report findings by sex. However, there are many biological questions related to brain health that go beyond sex differences and are indeed specific to women. Here, the focus is on why animal models should be utilized in the pursuit of understanding women's brain health, a brief overview of what they have provided thus far, and why they still hold tremendous promise. This review concludes with a set of suggestions for how to begin to pursue translational animal models in a way that facilitates rapid success and harnesses the most powerful aspects of animal models.

The promises and pitfalls of sex difference research.

Funding agencies in North America and Europe are recognizing the importance of the integration of sex differences into basic and clinical research. Although these mandates are in place to improve our knowledge of health for both men and women, there have been a number of implementation issues that require vigilance on the part of funders and the research community. Here we discuss issues on simple inclusion of both sexes in studies to specialisation of sex differences with attention paid to statistics and the need for sex-specific treatments. We suggest differing mandates need to be considered regarding simple integration versus the need for studies in the specialisation of sex differences and/or the need for research that recognises the importance of male-specific or female-specific factors that influence subsequent health such as menstruation, menopause or pregnancy.

What Does Sex Have to Do with It? The Role of Sex as a Biological Variable in the Development of Posttraumatic Stress Disorder.

PURPOSE OF REVIEW: This review highlights the neurobiological aspects of sex differences in posttraumatic stress disorder (PTSD), specifically focusing on the physiological responses to trauma and presents evidence supporting hormone and neurosteroid/peptide differences from both preclinical and clinical research. RECENT FINDINGS: While others have suggested that trauma type or acute emotional reaction are responsible for women's disproportionate risk to PTSD, neither of these explanations fully accounts for the sex differences in PTSD. Sex differences in brain neurocircuitry, anatomy, and neurobiological processes, such as those involved in learning and memory, are discussed as they have been implicated in risk and resilience for the development of PTSD. Gonadal and stress hormones have been found to modulate sex differences in the neurocircuitry and neurochemistry underlying fear learning and extinction. Preclinical research has not consistently controlled for hormonal and reproductive status of rodents nor have clinical studies consistently examined these factors as potential moderators of risk for PTSD. Sex as a biological variable (SABV) should be considered, in addition to the endocrine and reproductive status of participants, in all stress physiology and PTSD research.

Sex in Context: Limitations of Animal Studies for Addressing Human Sex/Gender Neurobehavioral Health Disparities.

Many brain and behavioral disorders differentially affect men and women. The new National Institutes of Health requirement to include both male and female animals in preclinical studies aims to address such health disparities, but we argue that the mandate is not the best solution to this problem. Sex differences are highly species-specific, tied to the mating system and social ecology of a given species or even strain of animal. In many cases, animals poorly replicate male-female differences in brain-related human diseases. Sex/gender disparities in human health have a strong sociocultural component that is intimately entangled with biological sex and challenging to model in animals. We support research that investigates sex-related variables in hypothesis-driven studies of animal brains and behavior. However, institutional policies that require sex analysis and give it special salience over other sources of biological variance can distort research. We caution that the costly imposition of sex analysis on nearly all animal research entrenches the presumption that human brain and behavioral differences are largely biological in origin and overlooks the potentially more powerful social, psychological, and cultural contributors to male-female neurobehavioral health gaps.

Studying both sexes: a guiding principle for biomedicine.

In May 2014, the U.S. National Institutes of Health (NIH) announced that it will ensure that investigators account for sex as a biological variable (SABV) in NIH-funded preclinical research as part of the agency's rigor and transparency initiative. Herein, I describe in more detail the rationale behind the SABV policy component and provide additional detail about policy goals. In short, studying both sexes is a guiding principle in biomedical research that will expand knowledge toward turning discovery into health. NIH expects that considering SABV in preclinical research will help to build a knowledge base that better informs the design of clinical research and trials in humans. Integrating the practice of studying both sexes in preclinical research will, over time, expand our currently incomplete knowledge base that plays a critical role in informing the development of sex- and gender-appropriate medical care for women and men.

Training in the implementation of sex and gender research policies: an evaluation of publicly available online courses.

BACKGROUND: Recently implemented research policies requiring the inclusion of females and males have created an urgent need for effective training in how to account for sex, and in some cases gender, in biomedical studies. METHODS: Here, we evaluated three sets of publicly available online training materials on this topic: (1) Integrating Sex & Gender in Health Research from the Canadian Institutes of Health Research (CIHR); (2) Sex as a Biological Variable: A Primer from the United States National Institutes of Health (NIH); and (3) The Sex and Gender Dimension in Biomedical Research, developed as part of "Leading Innovative measures to reach gender Balance in Research Activities" (LIBRA) from the European Commission. We reviewed each course with respect to their coverage of (1) What is required by the policy; (2) Rationale for the policy; (3) Handling of the concepts "sex" and "gender;" (4) Research design and analysis; and (5) Interpreting and reporting data. RESULTS: All three courses discussed the importance of including males and females to better generalize results, discover potential sex differences, and tailor treatments to men and women. The entangled nature of sex and gender, operationalization of sex, and potential downsides of focusing on sex more than other sources of variation were minimally discussed. Notably, all three courses explicitly endorsed invalid analytical approaches that produce bias toward false positive discoveries of difference. CONCLUSIONS: Our analysis suggests a need for revised or new training materials that incorporate four major topics: precise operationalization of sex, potential risks of over-emphasis on sex as a category, recognition of gender and sex as complex and entangled, and rigorous study design and data analysis.

Recently implemented research policies requiring the inclusion of females and males have created an urgent need for effective training in how to account for sex, and in some cases gender, in biomedical studies. We evaluated three publicly available online trainings on this topic: (1) Integrating Sex & Gender in Health Research from the Canadian Institutes of Health Research; (2) Sex as a Biological Variable: A Primer from the United States National Institutes of Health; and (3) The Sex and Gender Dimension in Biomedical Research, developed as part of "Leading Innovative Measures to Reach Gender Balance in Research Activities (LIBRA)" from the European Commission. We reviewed each course with respect to their coverage of (1) What is required by the policy; (2) Rationale for the policy; (3) Handling of the concepts "sex" and "gender;" (4) Research design and analysis; and (5) Interpreting and reporting data. All three discussed the importance of including males and females to better generalize results, discover potential sex differences, and tailor treatments to men and women. The interconnectedness of sex and gender, how to operationalize sex, and potential downsides of focusing on sex more than other sources of variation were minimally discussed. Notably, all three courses explicitly endorsed invalid analytical approaches that lead to incorrect findings of differences. Our analysis suggests a need for revised or new training materials that cover four major topics: precise operationalization of sex, attention to potential risks of over-emphasizing sex, consideration of gender and sex as complex and intertwined, and rigorous study design and data analysis.

The Promise and Practicality of Addressing Sex as a Biological Variable and the Ovarian Cycle in Preclinical Epilepsy Research.

Seizures and epilepsy affect people of all sexes and genders. In the last several years, funding agency initiatives such as the U.S. National Institutes of Health policy on sex as a biological variable (SABV) have intended to encourage researchers to study both males and females from cell to tissue to organism and analyze and report the resulting data with sex as a factor. Preclinical epilepsy research, however, continues to be plagued by confusion regarding both the SABV policy and its implementation, reflecting similar beliefs in the larger neuroscience research community. This article aims to address some common misconceptions and provide practical tools and suggestions for preclinical epilepsy researchers in implementing SABV and analysis of the female ovarian cycle (estrous cycle in rodents) in their research programs, with a focus on studies using rodent models. Examples of recent publications in preclinical epilepsy research highlighting the value of incorporating SABV and information on the estrous cycle are included. The specifics of how best to address SABV and the estrous cycle can vary depending on the needs and goals of a particular research program, but an embrace of these physiological factors by the preclinical epilepsy research community promises to yield more rigorous research and improved treatment strategies for all people with epilepsy.

mSphere of Influence: Venturing outside the biology canon with sex and gender.

Victoria Prieto-Echagüe works in the field of signaling by primary cilia, adipogenesis, and obesity. In this mShpere of Influence article, she reflects on how gender studies, feminism, and societal movements such as #metoo may inform all areas of biomedical and health research. She describes how they inspired her to incorporate sex as a biological variable (SABV) principle to her research exploring sex-specific mechanisms in obesity and metabolic diseases and argues that incorporating SABV is crucial for advancing precision medicine and addressing healthcare inequities.

Practical solutions for including sex as a biological variable (SABV) in preclinical neuropsychopharmacological research.

Recently, many funding agencies have released guidelines on the importance of considering sex as a biological variable (SABV) as an experimental factor, aiming to address sex differences and avoid possible sex biases to enhance the reproducibility and translational relevance of preclinical research. In neuroscience and pharmacology, the female sex is often omitted from experimental designs, with researchers generalizing male-driven outcomes to both sexes, risking a biased or limited understanding of disease mechanisms and thus potentially ineffective therapeutics. Herein, we describe key methodological aspects that should be considered when sex is factored into in vitro and in vivo experiments and provide practical knowledge for researchers to incorporate SABV into preclinical research. Both age and sex significantly influence biological and behavioral processes due to critical changes at different timepoints of development for males and females and due to hormonal fluctuations across the rodent lifespan. We show that including both sexes does not require larger sample sizes, and even if sex is included as an independent variable in the study design, a moderate increase in sample size is sufficient. Moreover, the importance of tracking hormone levels in both sexes and the differentiation between sex differences and sex-related strategy in behaviors are explained. Finally, the lack of robust data on how biological sex influences the pharmacokinetic (PK), pharmacodynamic (PD), or toxicological effects of various preclinically administered drugs to animals due to the exclusion of female animals is discussed, and methodological strategies to enhance the rigor and translational relevance of preclinical research are proposed.

Quantitative and qualitative sex difference in habenula-induced inhibition of midbrain dopamine neurons in the rat.

Introduction: Clinically relevant sex differences have been noted in a number of affective, behavioral, cognitive, and neurological health disorders. Midbrain dopamine neurons are implicated in several of these same disorders and consequently are under investigation for their potential role in the manifestation of these sex differences. The lateral habenula exerts significant inhibitory control over dopamine neuronal firing, yet little is known about sex differences in this particular neurocircuit. Methods: We performed in vivo, single unit, extracellular recordings of dopamine neurons in female and male anesthetized rats in response to single pulse stimulation of the lateral habenula. In addition, we assessed baseline firing properties of lateral habenula neurons and, by immunochemical means, assessed the distribution of estrogen receptor alpha cells in the lateral habenula. Results: Habenula-induced inhibition of dopamine neuronal firing is reduced in female rats relative to male rats. In addition, male rats had a higher prevalence of rebound excitation. Furthermore, the firing pattern of lateral habenula neurons was less variable in female rats, and female rats had a higher density of estrogen receptor alpha positive cells in the lateral habenula. Discussion: We found that the dopamine neuronal response to habenular stimulation is both qualitatively and quantitatively different in female and male rats. These novel findings together with reports in the contemporary literature lead us to posit that the sex difference in dopamine inhibition seen here relate to differential firing properties of lateral habenula neurons resulting from the presence of sex hormones. Further work is needed to test this hypothesis, which may have implications for understanding the etiology of several mental health disorders including depression, schizophrenia, and addiction.

Making a 'sex-difference fact': Ambien dosing at the interface of policy, regulation, women's health, and biology.

The U.S. Food and Drug Administration's (FDA) 2013 decision to lower recommended Ambien dosing for women has been widely cited as a hallmark example of the importance of sex differences in biomedicine. Using regulatory documents, scientific publications, and media coverage, this article analyzes the making of this highly influential and mobile 'sex-difference fact'. As we show, the FDA's decision was a contingent outcome of the drug approval process. Attending to how a contested sex-difference fact came to anchor elite women's health advocacy, this article excavates the role of regulatory processes, advocacy groups, and the media in producing perceptions of scientific agreement while foreclosing ongoing debate, ultimately enabling the stabilization of a binary, biological sex-difference fact and the distancing of this fact from its conditions of construction.

Toward a Systematic Assessment of Sex Differences in Cystic Fibrosis.

(1) Background: Cystic fibrosis (CF) is a disease with well-documented clinical differences between female and male patients. However, this gender gap is very poorly studied at the molecular level. (2) Methods: Expression differences in whole blood transcriptomics between female and male CF patients are analyzed in order to determine the pathways related to sex-biased genes and assess their potential influence on sex-specific effects in CF patients. (3) Results: We identify sex-biased genes in female and male CF patients and provide explanations for some sex-specific differences at the molecular level. (4) Conclusion: Genes in key pathways associated with CF are differentially expressed between sexes, and thus may account for the gender gap in morbidity and mortality in CF.

Sounding the Alarm: Sex Differences in Rat Ultrasonic Vocalizations during Pavlovian Fear Conditioning and Extinction.

Pavlovian fear conditioning is a prevalent tool in the study of aversive learning, which is a key component of stress-related psychiatric disorders. Adult rats can exhibit various threat-related behaviors, including freezing, motor responses, and ultrasonic vocalizations (USVs). While these responses can all signal aversion, we know little about how they relate to one another. Here we characterize USVs emitted by male and female rats during cued fear acquisition and extinction, and assess the relationship between different threat-related behaviors. We found that males consistently emitted >22 kHz calls (referred to here as "alarm calls") than females, and that alarm call frequency in males, but not females, related to the intensity of the shock stimulus. Interestingly, 25% of males and 45% of females did not emit any alarm calls at all. Males that did make alarm calls had significantly higher levels of freezing than males who did not, while no differences in freezing were observed between female Alarm callers and Non-alarm callers. Alarm call emission was also affected by the predictability of the shock; when unpaired from a tone cue, both males and females started emitting alarm calls significantly later. During extinction learning and retrieval sessions, males were again more likely than females to emit alarm calls, which followed an extinction-like reduction in frequency. Collectively these data suggest sex dependence in how behavioral readouts relate to innate and conditioned threat responses. Importantly, we suggest that the same behaviors can signal sex-dependent features of aversion.

Maxillary Sinus Dimensions in Skeletal Class I Chinese Population with Different Vertical Skeletal Patterns: A Cone-Beam Computed Tomography Study.

Due to the close relationship between the maxillary posterior teeth roots and the maxillary sinus floor, the maxillary sinus can significantly impact various dental treatments, including endodontic procedures and surgical apicectomy, periodontal flap surgery, surgical tooth extraction, dental implantation, and orthognathic surgeries. Specifically, in orthodontics, the location of the maxillary sinus floor may affect tooth movement and insertion of temporary anchorage devices (TADs). This study aims to evaluate the dimensions and location of the maxillary sinus in the Chinese orthodontic patient population with skeletal class I. Using cone-beam computed tomography (CBCT), the volumetric size, height, width, and depth of the sinus and the amount of alveolar bone below the sinus floor and buccal/palatal to the sinus wall were compared between patients of different genders and different vertical skeletal patterns. Unlike the previously reported skeletal class II population, the skeletal class I patients with different vertical patterns do not have significantly different size sinuses. On the other hand, males have larger maxillary sinuses in all parameters than females in the testing population. In addition, no significant correlation was noticed between the SN-MP angle and sinus dimensions or between the ANB angle and sinus dimensions. Nevertheless, the distance from the sinus floor to the alveolar bone crest is not correlated with skeletally sagittal or vertical parameters in females but negatively correlated with the skeletal sagittal parameter in males. In summary, different from the skeletal class II population, there is no significant difference in maxillary sinus size and location among different vertical skeletal patterns in the skeletal class I population. Compared to the skeletal class II population, a higher percentage of the skeletal class I population has an alveolar bone with less than 5 mm thickness, representing a narrowed safe zone of TADs placements.