Sex, Racial/Ethnic, and Regional Disparities in Pulmonary Embolism Mortality Trends in the USA, 1999-2020.

BACKGROUND: While the National Institutes of Health emphasize integrating sex as a biological variable into research, specific considerations of sex-related differences in pulmonary embolism (PE) mortality trends remain scarce. This study examines sex-based PE mortality trends across regional and demographic groups in the USA from 1999 to 2020. METHODS: A retrospective analysis of National Center for Health Statistics mortality data from 1999 to 2020 was conducted. Using ICD-10 code I26, PE decedents were identified. Piecewise linear regression assessed sex-based temporal trends in PE mortality by age, race/ethnicity, and census region. Annual percentage changes and average annual percentage changes were derived using Weighted Bayesian Information Criteria. The 95% confidence intervals were estimated using the empirical quantile method. RESULTS: From 1999 to 2020, a total of 179,273 individuals died in the USA due to PE, resulting in an age-adjusted mortality rate of 2.5 per 100,000 persons (95% CI, 2.5-2.5). While men and women exhibited comparable rates in recent time segments and across most subcategories, a higher mortality trend among males compared to females was observed among non-Hispanic White and Hispanic individuals and residents of the Western US census region. These results remained robust even after excluding data from 2020, accounting for the potential impact of the COVID-19 pandemic. CONCLUSIONS: Our study highlights sex-based disparities in PE mortality trends in the USA from 1999 to 2020. Despite overall stable mortality rates, higher trends among males were evident in specific demographic groups and regions. These findings emphasize the importance of targeted interventions to mitigate PE-related mortality discrepancies across diverse populations.

Perspective on statistical power and equivalence tests.

The use of both sexes or genders should be considered in experimental design, analysis, and reporting. Since there is no requirement to double the sample size or to have sufficient power to study sex differences, challenges for the statistical analysis can arise. In this article, we focus on the topics of statistical power and ways to increase this power. We also discuss the choice of an appropriate design and statistical method and include a separate section on equivalence tests needed to show the absence of a relevant difference.

Sexual dimorphisms in skeletal muscle: current concepts and research horizons.

The complex compositional and functional nature of skeletal muscle makes this organ an essential topic of study for biomedical researchers and clinicians. An additional layer of complexity is added with the consideration of sex as a biological variable. Recent research advances have revealed sexual dimorphisms in developmental biology, muscle homeostasis, adaptive responses, and disorders relating to skeletal muscle. Many of the observed sex differences have hormonal and molecular mechanistic underpinnings, whereas others have yet to be elucidated. Future research is needed to investigate the mechanisms dictating sex-based differences in the various aspects of skeletal muscle. As such, it is necessary that skeletal muscle biologists ensure that both female and male subjects are represented in biomedical and clinical studies to facilitate the successful testing and development of therapeutics for all patients.

Sex contextualism in laboratory research: Enhancing rigor and precision in the study of sex-related variables.

Understanding sex-related variation in health and illness requires rigorous and precise approaches to revealing underlying mechanisms. A first step is to recognize that sex is not in and of itself a causal mechanism; rather, it is a classification system comprising a set of categories, usually assigned according to a range of varying traits. Moving beyond sex as a system of classification to working with concrete and measurable sex-related variables is necessary for precision. Whether and how these sex-related variables matter-and what patterns of difference they contribute to-will vary in context-specific ways. Second, when researchers incorporate these sex-related variables into research designs, rigorous analytical methods are needed to allow strongly supported conclusions. Third, the interpretation and reporting of sex-related variation require care to ensure that basic and preclinical research advance health equity for all.

Developmental changes in lung function of mice are independent of sex as a biological variable.

Pulmonary function testing (PFT) in mice includes biomechanical assessment of lung function relevant to physiology in health and its alteration in disease, hence, it is frequently used in preclinical modeling of human lung pathologies. Despite numerous reports of PFT in mice of various ages, there is a lack of reference data for developing mice collected using consistent methods. Therefore, we profiled PFTs in male and female C57BL/6J mice from 2 to 23 wk of age, providing reference values for age- and sex-dependent changes in mouse lung biomechanics during development and young adulthood. Although males and females have similar weights at birth, females weigh significantly less than males after 5 wk of age (P < 0.001) with largest weight gain observed between 3 and 8 wk in females and 3 and 13 wk in males, after which weight continued to increase more slowly up to 23 wk of age. Lung function parameters including static compliance and inspiratory capacity also increased rapidly between 3 and 8 wk in female and male mice, with male mice having significantly greater static compliance and inspiratory capacity than female mice (P < 0.001). Although these parameters appear higher in males at a given age, allometric scaling showed that static compliance and inspiratory compliance were comparable between the two sexes. This suggests that differences in measurements of lung function are likely body weight-based rather than sex-based. We expect these data to facilitate future lung disease research by filling a critical knowledge gap in our field.NEW & NOTEWORTHY This study provides reference values for changes in mouse lung biomechanics from 2 to 23 wk of age. There are rapid developmental changes in lung structure and function of male and female mice between the ages of 3 and 8 wk. Male mice become noticeably heavier than female mice at or about 5 wk of age. We identified that differences in normal lung function measurements are likely weight-based, not sex-based.

Sex differences in the rodent medial prefrontal cortex - What Do and Don't we know?

The prefrontal cortex, particularly its medial subregions (mPFC), mediates critical functions such as executive control, behavioral inhibition, and memory formation, with relevance for everyday functioning and psychopathology. Despite broad characterization of the mPFC in multiple model organisms, the extent to which mPFC structure and function vary according to an individual's sex is unclear - a knowledge gap that can be attributed to a historical bias for male subjects in neuroscience research. Recent efforts to consider sex as a biological variable in basic science highlight the great need to close this gap. Here we review the knowns and unknowns about how rodents categorized as male or female compare in mPFC neuroanatomy, pharmacology, as well as in aversive, appetitive, and goal- or habit-directed behaviors that recruit the mPFC. We propose that long-standing dogmatic concepts of mPFC structure and function may not remain supported when we move beyond male-only studies, and that empirical challenges to these dogmas are warranted. Additionally, we note some common pitfalls in this work. Most preclinical studies operationalize sex as a binary categorization, and while this approach has furthered the inclusion of non-male rodents it is not as such generalizable to what we know of sex as a multidimensional, dynamic variable. Exploration of sex variability may uncover both sex differences and sex similarities, but care must be taken in their interpretation. Including females in preclinical research needs to go beyond the investigation of sex differences, improving our knowledge of how this brain region and its subregions mediate behavior and health. This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".

The role of neurosteroids in posttraumatic stress disorder and alcohol use disorder: A review of 10 years of clinical literature and treatment implications.

Rates of alcohol use disorder (AUD) are increasing in men and women and there are high rates of concurrent posttraumatic stress disorder (PTSD) and AUD. AUD and PTSD synergistically increase symptomatology and negatively affect treatment outcomes; however, there are very limited pharmacological treatments for PTSD/AUD. Neurosteroids have been implicated in the underlying neurobiological mechanisms of both PTSD and AUD and may be a target for treatment development. This review details the past ten years of research on pregnenolone, progesterone, allopregnanolone, pregnanolone, estradiol, testosterone and dehydroepiandrosterone/dehydroepiandrosterone-sulfate (DHEA/DHEA-S) in the context of PTSD and AUD, including examination of trauma/alcohol-related variables, such as stress-reactivity. Emerging evidence that exogenous pregnenolone, progesterone, and allopregnanolone may be promising, novel interventions is also discussed. Specific emphasis is placed on examining the application of sex as a biological variable in this body of literature, given that women are more susceptible to both PTSD diagnoses and stress-related alcohol consumption.

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.

Best practices to promote rigor and reproducibility in the era of sex-inclusive research.

To enhance inclusivity and rigor, many funding agencies and journals now mandate the inclusion of females as well as males in biomedical studies. These mandates have enhanced generalizability and created unprecedented opportunities to discover sex differences. However, education in sound methods to consider sex as a subgroup category has lagged behind, resulting in a problematic literature in which study designs, analyses, and interpretations of results are often flawed. Here, we outline best practices for complying with sex-inclusive mandates, both for studies in which sex differences are a primary focus and for those in which they are not. Our recommendations are organized within the "4 Cs of Studying Sex to Strengthen Science: Consideration, Collection, Characterization and Communication," a framework developed by the Office of Research on Women's Health at the National Institutes of Health in the United States. Following these guidelines should help researchers include females and males in their studies while at the same time upholding high standards of rigor.

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.

Lower female survival from an opportunistic infection reveals progesterone-driven sex bias in trained immunity.

Immune responses differ between females and males, although such sex-based variance is incompletely understood. Observing that bacteremia of the opportunistic pathogen Burkholderia gladioli caused many more deaths of female than male mice bearing genetic deficiencies in adaptive immunity, we determined that this was associated with sex bias in the innate immune memory response called trained immunity. Female attenuation of trained immunity varies with estrous cycle stage and correlates with serum progesterone, a hormone that decreases glycolytic capacity and recall cytokine secretion induced by antigen non-specific stimuli. Progesterone receptor antagonism rescues female trained immune responses and survival from controlled B. gladioli infection to magnitudes similar to those of males. These data demonstrate progesterone-dependent sex bias in trained immunity where attenuation of female responses is associated with survival outcomes from opportunistic infection.

Developing Researchers with Expertise in Sex as a Biological Variable through SCORE Career Enhancement Core Center Programs.

There is a critical need for interdisciplinary and translational scientists to apply sex as a biological variable (SABV) research to address knowledge gaps in the health of women. In 2018, the Office of Research on Women's Health (ORWH) partnered with several National Institute of Health (NIH) Institutes and Centers to expand the Specialized Centers of Research (SCOR) Excellence (SCORE) Programs (together referred to as SCOR/E) with an important feature-the Career Enhancement Core (CEC). The SCORE CEC mentors early career investigators to become the next generation of biomedical and behavioral researchers focused on SABV and women's health. In this article, we outline our approach at the Yale University SCORE to support early career trajectories through the provision of salary support, educational curricula, translational mentorship, pilot project funding, and professional development. Using the Yale-SCOR/E CEC Programs as instructional models, we highlight critical measures of academic success, namely grant funding and publications, among early career investigators. At Yale University, 12 pilot projects funded by the SCOR/E Programs resulted in 14 extramural grants, amounting to an $80 return on every $1 invested in "seed" funding. So far, our SCOR/E Programs have resulted in 129 publications, 83% of which were first-authored by trainees, and 100% of trainees continued research careers with an emphasis on SABV. Finally, we provide recommendations on how biomedical scientists can apply SABV in their studies of major medical conditions in an interdisciplinary and integrative way.

Microvascular Aging and Eicosanoids-Women's Evaluation of Systemic Aging Tenacity ("You are never too old to become younger!") Specialized Center of Research Excellence on Sex Differences Career Enhancement Core: Building the Future.

The objective of the National Institutes of Health Office of Research in Women's Health (NIH/ORWH) Specialized Center of Research and Career Enhancement (SCORE) program is to expedite the development and application of new knowledge that affect women, to learn more about the etiology of these diseases, and to foster improved approaches to treatment and/or prevention. Each SCORE has a Career Enhancement Core (CEC) that serves to meet the career enhancement needs of translational science in the study of sex differences. The Microvascular Aging and Eicosanoids-Women's Evaluation of Systemic aging Tenacity (MAE-WEST) ("You are never too old to become younger!") Specialized Center of Research Excellence (SCORE) on Sex Differences will study pro- and anti-inflammatory responses and small vessel aging traits. As part of our SCORE CEC, we have advanced several initiatives to embed consideration of sex as a biological variable (SABV) into the infrastructure of our two CEC institutions. Unlike other professions, ongoing physician education through continuing medical education (CME) activities is required and embedded in the practice of medicine. The MAE-WEST SCORE in collaboration with the CSMC Clinical Scholars Program, the Center for Research in Women's Health and Sex-differences and the CSMC CME Office requires SABV and as Diversity, Equity, and Inclusion components in all CSMC CME programs. Clinical practice is also increasingly guided by evidence-based guidelines, with Class I recommendations resulting from clinical trials rather than expert consensus. It is essential that women be included in clinical trials proportionate to the prevalence and burden of disease. The MAE-WEST SCORE has developed our own unique CEC for providing novel educational, networking, funding opportunities, and translation to practice support. The developed best practices have found novel ways to enhance studies of women's health and SABV. We welcome visitors on-site and virtual to share with the broader academic and practicing community.

Establishment of a Private Foundation-Academic Partnership to Promote Careers of Early-Stage Investigators Examining the Influence of Sex and Gender on Health and Health Care.

Biological sex and gender-based constructs contribute significantly to the diversity of disease outcomes and treatment responses across the life course. To promote research considering sex and gender, the National Institutes of Health (NIH) Office of Research on Women's Health (ORWH) launched the Specialized Centers of Research Excellence (SCORE) on sex differences program. The Career Enhancement Core (CEC) of the Johns Hopkins SCORE on Sex and Age Differences in Immunity to Influenza (SADII) partnered with the Foundation for Gender-Specific Medicine, which matched NIH funding to support seed grants. Over 3 years we awarded 12 (10 were women faculty) seed grants to early-stage investigators. One year after the award, the seed grant awardees highlighted their progress, including publications, grant applications, and abstracts. All awardees noted challenges with their progress related to the COVID-19 pandemic and supply chain delays and shared suggestions for improving the programming of the CEC. They also highlighted the multiple ways the awards had helped them gain pilot data toward larger grants, build collaborative relationships, and present at the annual SCORE symposium. We describe a model and evidence supporting a private-academic collaboration to support the careers of early-stage investigators conducting research related to sex and gender.

Considering Sex as a Variable at a Research University: Knowledge, Attitudes, and Practices.

Biomedical research has a history of excluding females as research subjects, which threatens rigor, reproducibility, and inclusivity. In 2016, to redress this bias, the U.S. National Institutes of Health (NIH) implemented a policy requiring the consideration of sex as a biological variable (SABV) in all studies involving vertebrate animals, including humans. Unless strongly justified, females and males must be included in all studies and results reported disaggregated by sex. Recent evidence indicates, however, that misunderstandings of the policy and other significant barriers impede its implementation. To shed light on those barriers at our home institution, we conducted a study funded by the Emory University Specialized Center of Research Excellence on Sex Differences (SCORE). In semistructured interviews of Emory principal investigators in the biological sciences, we noted their knowledge of what the policy entails and why it was implemented, their attitudes toward it, and the extent to which it has or has not changed their research practices. Although attitudes toward SABV were generally positive, most researchers face challenges with respect to its implementation. We suggest interventions that can be mounted at the level of home institutions, such as raising awareness of locally available core facilities, to help address these challenges. More training is needed on what the policy asks of researchers, how sex is defined, the nonhormonal ways that sex differences can manifest, and best practices for statistical analysis of sex-based data. Home institutions may also want to explore ways to lessen the stress associated with rollout of SABV policy.

Inclusion of Sex as a Biological Variable in Biomedical Sciences at the Undergraduate Level and Beyond.

To improve research on women's health, and to achieve better understanding of the factors controlling disease across diverse populations of humans, it is imperative to study sex differences in physiology and disease. After the introduction of the "SABV policy" at NIH, which requires investigators using animals or humans to consider sex as a biological factor, it became clear that many investigators were unaware of concepts of sexual differentiation or methods that can be used to study sex as a biological variable (SABV). To remedy this situation, efforts have increased to teach concepts and methods of SABV at all educational levels. The UCLA Scientific Center of Research Excellence (SCORE) grant "Sex differences in the metabolic syndrome" promotes education about SABV through three primary mechanisms: (1) through didactic course content for students at the undergraduate level, (2) by providing pilot funding for early career investigators to study the role of sex in metabolism-related areas, and (3) through curation of a video library, which may be useful for investigators performing research at the graduate, postgraduate, and faculty levels.

Pharmacogenetics of sodium-glucose co-transporter-2 inhibitors: Validation of a sex-agnostic pharmacodynamic biomarker.

AIM: To validate pharmacodynamic responses to sodium-glucose co-transporter-2 (SGLT2) inhibitors and test for association with genetic variants in SLC5A4, SLC5A9, and SLC2A9. METHODS: Canagliflozin (300 mg), a SGLT2 inhibitor, was administered to 30 healthy volunteers. Several endpoints were measured to assess clinically relevant responses, including drug-induced increases in urinary excretion of glucose, sodium and uric acid. RESULTS: This pilot study confirmed that canagliflozin (300 mg) triggered acute changes in mean levels of several biomarkers: fasting plasma glucose (-4.1 mg/dL; P = 6 × 10-5 ), serum creatinine (+0.05 mg/dL; P = 8 × 10-4 ) and serum uric acid (-0.90 mg/dL; P = 5 × 10-10 ). The effects of sex on glucosuria depended upon how data were normalized. Whereas males' responses were ~60% greater when data were normalized to body surface area, males and females exhibited similar responses when glucosuria was expressed as grams of urinary glucose per gram-creatinine. The magnitude of glucosuria was not significantly correlated with fasting plasma glucose, estimated glomerular filtration rate or age in those healthy individuals without diabetes with an estimated glomerular filtration rate of more than 60 mL/min/1.73m2 . CONCLUSIONS: Normalizing data relative to creatinine excretion will facilitate including data from males and females in a single analysis. Furthermore, because our ongoing pharmacogenomic study (NCT02891954) is conducted in healthy individuals, this will facilitate detection of genetic associations with limited confounding by other factors such as HbA1c and renal function.

Reasons for the Sex Bias in Osteoarthritis Research: A Review of Preclinical Studies.

Osteoarthritis (OA) is one of the most common degenerative diseases of articular cartilage. During OA, all the elements that contribute to the joint undergo physiological and structural changes that impair the joint function and cause joint pain and stiffness. OA can arise naturally, with the aging population witnessing an increase in diagnoses of this pathology, but the root causes of OA have yet to be identified, and increasing interest is arising towards investigating biological sex as a risk factor. Clinical studies show increased prevalence and worse clinical outcomes for female patients, yet most clinical and preclinical studies have disproportionately focused on male subjects. This review provides a critical overview of preclinical practices in the context of OA, highlighting the underlying need for taking biological sex as both a risk factor and an important component affecting treatment outcome. A unique insight into the possible reasons for female underrepresentation in preclinical studies is offered, including factors such as lack of specific guidelines requiring the analysis of sex as a biological variable (SABV), research-associated costs and animal handling, and wrongful application of the reduction principle. Additionally, a thorough investigation of sex-related variables is provided, stressing how each of them could add valuable information for the understanding of OA pathophysiology, as well as sex-dependent treatment strategies.

Quality control and analytic best practices for testing genetic models of sex differences in large populations.

Phenotypic sex-based differences exist for many complex traits. In other cases, phenotypes may be similar, but underlying biology may vary. Thus, sex-aware genetic analyses are becoming increasingly important for understanding the mechanisms driving these differences. To this end, we provide a guide outlining the current best practices for testing various models of sex-dependent genetic effects in complex traits and disease conditions, noting that this is an evolving field. Insights from sex-aware analyses will not only teach us about the biology of complex traits but also aid in achieving the goals of precision medicine and health equity for all.

Timing matters: Sex differences in inflammatory and behavioral outcomes following repetitive blast mild traumatic brain injury.

BACKGROUND: Repetitive blast-related mild traumatic brain injury (mTBI) caused by exposure to high explosives is increasingly common among warfighters as well as civilians. While women have been serving in military positions with increased risk of blast exposure since 2016, there are few published reports examining sex as a biological variable in models of blast mTBI, greatly limiting diagnosis and treatment capabilities. As such, here we examined outcomes of repetitive blast trauma in female and male mice in relation to potential behavioral, inflammatory, microbiome, and vascular dysfunction at multiple timepoints. METHODS: In this study we utilized a well-established blast overpressure model to induce repetitive (3x) blast-mTBI in both female and male mice. Acutely following repetitive exposure, we measured serum and brain cytokine levels, blood-brain barrier (BBB) disruption, fecal microbial abundance, and locomotion and anxiety-like behavior in the open field assay. At the one-month timepoint, in female and male mice we assessed behavioral correlates of mTBI and PTSD-related symptoms commonly reported by Veterans with a history of blast-mTBI using the elevated zero maze, acoustic startle, and conditioned odorant aversion paradigms. RESULTS: Repetitive blast exposure resulted in both similar (e.g., increased IL-6), and disparate (e.g., IL-10 increase only in females) patterns of acute serum and brain cytokine as well as gut microbiome changes in female and male mice. Acute BBB disruption following repetitive blast exposure was apparent in both sexes. While female and male blast mice both exhibited acute locomotor and anxiety-like deficits in the open field assay, only male mice exhibited adverse behavioral outcomes that lasted at least one-month. DISCUSSION: Representing a novel survey of potential sex differences following repetitive blast trauma, our results demonstrate unique similar yet divergent patterns of blast-induced dysfunction in female vs. male mice and highlight novel targets for future diagnosis and therapeutic development.