This article has been cited by other articles in PMC. Abstract Background Cardiovascular disease remains the primary cause of death worldwide. In the US, deaths due to cardiovascular disease for women exceed those of men. While cultural and psychosocial factors such as education, economic status, marital status and access to healthcare contribute to sex differences in adverse outcomes, physiological and molecular bases of differences between women and men that contribute to development of cardiovascular disease and response to therapy remain underexplored.
Determining whether a sex difference exists in a trait must take into account the reproductive status and history of the animal including those used for tissue cell harvest, such as the presence of gonadal steroids at the time of testing, during development or number of pregnancies. When selecting the type of experimental animal, additional consideration should be given to diet requirements soy or plant based influencing consumption of phytoestrogen , lifespan, frequency of estrous cycle in females, and ability to investigate developmental or environmental components of disease modulation.
Care must be given to selection of hormonal treatment and route of administration. Conclusions Accounting for sex in the design and interpretation of studies including pharmacological effects of drugs is essential to increase the foundation of basic knowledge upon which to build translational approaches to prevent, diagnose and treat cardiovascular diseases in humans.
Thus, there is a need for improvement in the current preventive, diagnostic and treatment strategies by accounting for sex-specific differences in the etiology and risk factors of cardiovascular disease. In , the Institute of Medicine advocated that a better understanding of differences in human diseases between males and females, with translation of these differences into clinical practice, requires consideration of sex as an important biological variable in design of basic research [ 5 ].
Funding agencies in the US, Canada and the European Union require inclusion of women in governmental sponsored research and analyses of outcomes by sex [ 6 - 10 ]. Often, however, implementation of those requirements is not met.
Even if the requirement is met, including men and women in clinical studies often does not advance the goal of understanding sex differences as there is no requirement to compare the sexes nor is there a requirement to sufficiently power studies to do so [ 11 - 15 ]. Furthermore, there are no requirements for inclusion of male and female animals in basic, mechanistic, or preclinical studies and often sex is not reported as a critical biological variable in the study design [ 16 - 18 ].
This paper is intended as a guide to help formulate hypothesis-driven studies of cardiovascular function accounting for sex as a biological variable. Design considerations for studies on sex differences of cardiovascular disease will be discussed to extend those which have been developed for studies of brain and behavior [ 19 , 20 ], neuroprotection after stroke [ 21 ], and pain and analgesia [ 22 ].
Questions are posed to direct investigators toward optimizing the choice of experimental system to maximize the information gained for translation to human medicine.
Application of genetic studies including quantitative trait locus mapping will be evaluated in relationship to cardiovascular phenotypes of different model species. Finally, recommendations are provided for statistical analysis and future research directions. Accounting for sex in the design and interpretation of basic research of cardiovascular structure, function and disease is essential to achieve scientific excellence whether such studies utilize cultured cells, isolated tissues, or experimental animals [ 23 , 24 ] and is essential to increase the foundation of basic knowledge upon which to build translational approaches to medical care of humans [ 25 - 28 ].
Information provided in this paper will help guide the investigator toward that excellence. Definitions Sex, a biological construct, gender, a psychosocial construct, and environment contribute to disparities in cardiovascular disease separately or through interactions.
Studies using cells and tissues or experimental animals can be designed to identify these differences and interactions. Terminology related to sex steroid hormones 'Sex', a biological construct, refers to biological differences defined by sex chromosomes XX, XY and the presence of functional reproductive organs and sex steroids [ 5 , 19 , 20 ]. Biological questions require identification of which cells, tissues and organs demonstrate dimorphic structure and function related to cardiovascular disease.
Thus, gender can influence biological outcomes. Thus, sex is considered a dichotomous variable; gender is a continuous variable as defined by a range of characteristics that might vary with age, species animals , or ethnicity humans , geographical location, education, and culture. Most studies using animals categorized by anatomical features and chromosomes can be described as studies of 'sex differences', with identification of dimorphic cellular, tissue, or organ functions.
However, studies using animals can also be designed to answer questions which address psychosocial constructs, such as whether hierarchical social interactions are biologically based and whether they influence biological imprinting, or vice versa, in relationship to cardiovascular disease.
What are sex steroid hormones? As described above, sex is defined by the XX or XY chromosomal complement and the presence of sex organs and sex steroid hormones. Sex steroid hormones fall into three general classes of hormones: Intracellular and intranuclear actions of hormones belonging to these classes are mediated through binding of the hormones to specific receptors.
Extensive reviews of the types and cellular mechanisms of action of these receptors are available [ 29 - 36 ]. Designing experiments in which one intends to evaluate effects of sex steroids on cardiovascular disease processes require the mention of certain caveats. First, hormones belonging to each of these classes are synthesized naturally in mammals. However, synthetic analogs of the naturally produced hormones are also available commercially. Most natural steroid hormones bind to only one class of intracellular receptor.
However, many synthetic steroids bind to multiple classes of steroid receptors. In addition, certain progestogens may bind to the androgen receptor. These may antagonize actions of endogenous androgens by reversibly binding to the androgen receptor but because these progestogens are 'weak androgens', their binding does not initiate the full cascade of intracellular actions such as translocation to the nucleus and initiation receptor-mediated DNA transcription [ 39 - 41 ].
This issue is of particular importance in evaluating effects of hormonal treatments hormonally based contraception, oophorectomy and hormonal treatments, including those of selective estrogen receptor modulators SERMS on cardiovascular function and disease. A second consideration regarding studies utilizing sex steroid hormones is that of tissue production independent of primary sources. Although the primary source of production of androgens is the testes in males and the ovary for production of estrogens and progesterone in females, local steroidogenesis and possible production of steroids from 'precursor' compounds at the cell membrane or within the cytoplasm in non-sex organs may also occur.
Thus, steroid exposure may occur on a tissue or cell level that is not reflected by measurement of circulating levels of sex steroids [ 42 - 46 ]. The third consideration regarding the evaluation of hormonal effects in design of studies investigating sex differences is that metabolic products of androgens and estrogens have biologic activity. The androgen, 5-dehydroepiandrosterone DHEA is synthesized from cholesterol in the adrenal gland, testes and ovaries.
Testosterone, as well as five other androgens, is synthesized from DHEA. Therefore, for studies in which the investigator administers an androgen but does not want to risk increasing estradiol synthesis, DHT can be substituted for testosterone.
However, DHT is more biologically active because it binds to the androgen receptor with a fold higher affinity than testosterone [ 31 ]. The adrenal glands also manufacture and secrete estradiol and estrone. Oral products through the 'first pass effect' also increase production of liver proteins, such as sex hormone-binding globulin SHBG and clotting factors, with attendant biological consequences.
Terminology is also important. For example, a mixture of conjugated estrogen metabolites derived from urine of pregnant mares conjugated equine estrogen CEE , which contains primarily equilin sulfate and estrone sulfate and other metabolites of estrogen, as well as androgens was used in the Women's Health Initiative. Oral administration of this compound was used to determine effects of 'estrogen treatment' for primary prevention of cardiovascular disease in postmenopausal women.
Origins of sex differences All biological sex differences are initiated by the genes of the sex chromosomes, which are the only genes that are inherited in a sex-specific manner. Differences between XY and XX cells can be attributed to: The SRY gene on the Y chromosome, which determines the development of the testis and the subsequent secretion of male sex hormones, also influences expression of other genes on the autosomal chromosomes.
Sex hormones at critical periods in development influence cellular differentiation, which may also be influenced by environmental factors. Another way to frame these interactions is as hormone-independent sexual differentiation, hormone-dependent sexual differentiation and sex-specific hormone actions.
Determining whether a trait or condition is a sex difference requires a systematic approach to separate the interactions of sex chromosomes, sex hormones and environment. A logical series of experimental questions that has been proposed for studies in brain and behavior [ 19 ] can serve as a guide for investigators in cardiovascular disease research as well.
And 6 does the sex difference result from a sex chromosomal influence on autosomal gene expression? Choice of experimental systems The choice of an experimental system will depend on the question being asked and the hypothesis to be tested.
In contemporary publishing, methodological details including the sex of the research material or animals are often omitted from research papers or relegated to supplementary material. However, it is just these methodological details that allow investigators to reproduce experiments of others and to understand how important variables such as sex or hormonal status may influence outcomes. The Institute for Laboratory Animal Research and their official publication ILAR Journal is a rich resource for details regarding reproductive, hormonal and developmental information of various animal species used in cardiovascular disease research.
Several general considerations are provided below for some of the more common experimental systems employed to address mechanisms of cardiovascular function. Cells in culture and isolated tissues Cultured vascular endothelial cells, smooth muscle cells and cardiac myocytes including neonatal cells , as well as isolated blood vessels and hearts, are used in experiments that explore intracellular signaling mechanisms in the development and treatment of cardiovascular disease.
The same can be said for stem and progenitor cells being cultured for cell-based therapies [ 63 - 66 ]. For example in mice, cells derived from female animals appear to be more effective at both reversing disease and restoring a pre-disease level of cells to bone marrow than male-derived cells [ 66 ]. Differences in the efficacy of male and female cells could possibly be attributed to differences in paracrine factors for example, cytokines [ 65 ]. Comparison of these preclinical vascular data to early human clinical data suggest that sex-based differences in progenitor cell numbers and function seen in mouse models of atherosclerosis and acute myocardial infarction reflect human clinical scenarios [ 64 ].
The following points should be addressed in design of experiments using cultured cells, isolated stem and progenitor cells, and isolated tissues: If so, is the donor appropriate for the mechanism of interest related to human disease in regard to sex, age and hormonal status?
This method has been refined and extended to a high-throughput automated setting [ 68 ]. Sex of neonatal animals including mice and rats can be determined by examining the anogenital distance [ 69 ].
In larger animals the presence of gonads should be confirmed as some male animals obtained from commercial suppliers in particular, pigs may be castrated at birth, and, therefore, studies in these animals would yield cells developed or differentiated under a sex hormone-depleted environment. As sex differences are influenced both by the sex chromosomes and sex hormones, care must be taken to control the hormonal environment of cultured cells.
Media can be stripped of hormones by charcoal treatment. Thus, a given concentration of sex steroid added at day 1 of a culture cycle may not be sustained over a long period of time. Alternatively, exposure of cells to hormones may 'imprint' the cell phenotype over several passages [ 70 , 71 ]. As sex steroid hormones initiate rapid actions that do not require gene transcription non-genomic actions as well as effects on gene transcription genomic actions that represent different temporal sequences, duration of exposure to the hormone of interest is a critical consideration in study design.
However, estradiol also acutely increases eNOS activity and release of nitric oxide via a non-genomic effect that is due to an increase in intracellular calcium [ 37 ]. However, the specificity of this receptor is controversial since GPR30 also mediates rapid aldosterone-mediated effects on the vasculature [ 32 , 33 , 72 ].
Androgens also produce genomic effects via androgen response elements AREs in genes, but cause acute vasodilation by a non-genomic mechanism that involves activation of calcium activated potassium channels [ 73 , 74 ]. Future studies are necessary to completely understand the genomic and non-genomic effects of sex steroids and the mechanisms responsible for modulating cardiovascular function including whether the chromosomal constitution of the cell modulates responses to steroids via transcription factors, differential expression of protein chaperones, methylation of DNA, and so on [ 32 , 33 , 72 ].
Experimental animals Several mammals are used in studies of cardiovascular disease. Of the small mammals, rats and mice are used most often to model cardiovascular disease because of their short life span, short estrous cycle and gestation, modest cost of housing, and the ability to perform genetic manipulation in them. Both the strain and sex of the animals are known to influence expression of cardiovascular pathologies [ 79 , 80 ].
Transgenic mice and some transgenic rats that contain estrogen and androgen receptor knockouts are commercially available for study. Although preliminary, use of these animals to study the onset and progression of vascular disease has shown that the number of vascular progenitor cells found in bone marrow and blood is decreased in estrogen receptor knockout ERKO mice compared to wild type controls.
These transgenic animals are especially valuable to study the role of sex steroids and their receptors in cardiovascular disease. For example, the global ARKO exhibit neutropenia, osteopenia, low levels of serum testosterone, arrested spermatogenesis, and females have a reduced number of pups compared to wild type mice [ 81 ]. Therefore, especially for androgen receptor, specific tissue-directed androgen receptor knockouts, using Cre-loxP methods, are preferred [ 82 - 85 ].
Cre-loxP technology allows for knockout or expression of a gene of interest in a specific tissue [ 86 ].
These loxP sites contain two 13 bp inverted repeats surrounding an 8 bp core sequence that provides directionality. Thus, two transgenic mouse strains are necessary to develop a cell-specific knockout: Following one round of crossbreeding, double transgenic pups containing both the floxed transgene and the Cre transgene are selected by genotyping.