Sexual dimorphism in biomedical research: a call to analyse by sex

Despite the huge body of evidence that males and females have very different immune systems and respond differently to immune challenge, few biomedical studies consider sex in their analyses. This editorial discusses the underlying biology behind immunological sex differences, their effects on immunity to infections and vaccines, and the reasons why sex differences are frequently overlooked in biomedical research. Readers are urged to design their future studies in order to analyse by sex, and to analyse existing datasets by sex. The information gained is likely to be of considerable importance in our current understanding of immune mechanisms. Sex refers to the intrinsic characteristics that distinguish males from females, whereas gender refers to the socially determined behaviour, roles or activities that males and females adopt. Male and female immune systems are not equal, leading to clear sexual dimorphism in response to infections and vaccination. In 2010, Nature featured a series of articles aimed at raising awareness regarding the inherent sex bias in modern day biomedical research and yet little has changed since that time.1–3 They comment that modern day medical practice is less evidence-based for women than for men due to a sex bias towards the study of males in biomedical research. They further suggest that journals and funders should insist on studies being conducted in both sexes, or that authors should state the sex of animals used in their studies. Unfortunately this was not widely adopted. This editorial will discuss the literature regarding sex differences in immunity to infections and vaccines and urge the readership to consider sex in their future biomedical studies. Even before they are born, intrauterine differences begin to differentially shape male and female immune systems. The male intrauterine environment is more inflammatory than that of females,4 male fetuses produce more androgens5 and have higher IgE levels,6 all of which lead to sexual dimorphism before birth. Furthermore, male fetuses have been shown to undergo more epigenetic changes than females with decreased methylation of many immune response genes, probably due to physiological differences.7 The X chromosome contains numerous immune response genes, such as toll-like receptors 7 and 8, multiple cytokine receptors, genes involved in T cell and B cell activity, and transcriptional and translational regulatory factors;8 while the Y chromosome encodes for a number of inflammatory pathway genes that can only be expressed in males.9 Females have two X chromosomes, one of which is inactivated, usually leading to expression of the wild type gene. X inactivation is incomplete or variable,10 which is thought to contribute to greater inflammatory responses among females.11,12 The immunological X and Y chromosome effects will begin to manifest in utero leading the sex differences in immunity from birth, which continue throughout life. MicroRNAs (miRNAs) regulate physiological processes, including cell growth, differentiation, metabolism and apoptosis.13 Males and females differ in their miRNA expression,14 even in embryonic stem cells;15 oestrogens16 and testosterone17 can regulate miRNAs expression, although testosterone is thought to work via its conversion to oestradiol;18 and the X chromosome is particularly enriched in miRNAs involved in immunity.13 All these sex-differential miRNA factors likely contribute to sex differences in the prevalence, pathogenesis and outcome of infections and vaccination. Females are born with higher oestriol concentrations than males, while males have more testosterone.19 Shortly after birth, male infants undergo a transient activation of the hypothalamo-pituitary-gonadal axis, characterised by a testosterone surge, while the female effect is variable. This so called ‘mini-puberty’ peaks at approximately 3 months of age.20 Once puberty begins, the ovarian hormones such as oestrogen dominate in females, while testicular-derived androgens dominate in males. Many immune cells express sex hormone receptors, allowing the sex hormones to influence immunity. Very broadly, oestrogens are Th2 biasing and pro-inflammatory,21,22 whereas testosterone is Th1 skewing and immunosuppressive.23–26 Thus sex steroids undoubtedly play a major role in sexual dimorphism in immunity throughout life. Numerous sex differences in susceptibility to infections have been described in the literature. Males fare worse during sepsis;27–29 they are more susceptible to certain bacterial infections such as invasive pneumococcal disease,30 group A streptococcal pharyngitis,31 and enterohaemorrhagic E. coli31 and campylobacter diarrhoea;32 and pulmonary tuberculosis is more prevalent among adult males.33 By contrast, females suffer more Mycoplasma pneumoniae and Bordetella pertussis infections.31 In general, viral infections are more prevalent among males, while the clinical outcomes are often more severe among females due to a more pronounced immune response leading to increased immunopathology.34 A greater male susceptibility to viral infections in childhood may reverse to a female preponderance in adulthood,31 although females are more susceptible to certain viruses such as herpes infections from childhood.35 Males are generally more susceptible to parasitic infections, including Leishmania spp., Plasmodium falciparum and P. vivax, Schistosoma mansoni and certain filarial infections.36 Females seem more susceptible to Toxoplasma gondii infection36 and possibly to Giardia lamblia and Entamoeba histolytica.37 The protective effects of breast feeding may not be the same in males and females, since breast feeding protects females but not males against severe acute lung disease.38 These effects cannot simply be explained by gender differences, but seem to be true effects of sex. Sex differences have been described for almost every commercially available vaccine in use.39 Females mount higher antibody responses to certain vaccines such as measles, hepatitis B, influenza and tetanus vaccines, while males have better antibody responses to yellow fever, pneumococcal polysaccharide and meningococcal A and C vaccines.40 However, the data are conflicting with some studies showing sex effects, whereas other studies show none. Post-vaccination clinical attack rates also vary by sex with females experiencing less influenza41,42 and males experiencing less pneumococcal disease after vaccination.43 Females experience more adverse events (AEs) to certain vaccines, such as oral polio vaccine44 and influenza vaccine,45 while males have more AEs to other vaccines such as yellow fever vaccine,40,46 suggesting the sex effect varies according to the vaccine given. The existing data hint at higher vaccine-related AEs in infant males progressing to a female preponderance from adolescence, suggesting a hormonal effect, but this has not been confirmed in a systematic review. If male and female immune systems behave in opposing directions with one pro-inflammatory/Th2 and one anti-inflammatory/Th1 , then clearly analysing them together may well cause effects and responses to be cancelled out. Separate analysis by sex would detect effects that were not seen in the combined analysis. Furthermore, a dominant effect in one of the sexes might be wrongly attributed to both sexes. For drug and vaccine trials this could have serious implications. Given the huge body of evidence that males and females are immunologically divergent, why do most scientific studies fail to analyse by sex? Traditionally in science the sexes have been regarded as being equal and the main concern has been to recruit an equivalent number of males and females into studies. Adult females are often not enrolled into drug and vaccine trials because of the potential interference of hormones of the menstrual cycle or risk of pregnancy; thus, most data come from trials conducted in males only. Similarly, the majority of animal studies are conducted in males, although many animal studies fail to disclose the sex of the animals used.2 Analysing data by sex adds the major disadvantage that sample sizes would need to double in order to have sufficient power to detect significant sex effects. This potentially means double the cost and double the time to conduct the study, in a time when research funding is limited and hard to obtain. Furthermore, since the funders don't request analysis by sex, and the journals do not ask for it, it is not a major priority in today's highly competitive research environment. It is likely that we are missing important scientific information by not investigating more comprehensively how males and females differ in immunological and clinical trials. We are entering an era in which there is increasing discussion regarding personalised medicine. Therefore, it is quite reasonable to imagine that females and males might benefit differently from certain interventions such as vaccines, immunotherapies and drugs. The mindset of the scientific community needs to shift. We are in an era where we can analyse the microbiome in various body compartments,47 can analyse the genome and every gene transcribed by it,48 we can analyse the epigenome,49 the metabolome,50 the proteome,51 and multiple cytokines and chemokines in a single small sample,52 and yet we still fail to allow sufficient sample size to do a separate analysis by sex. I appeal to readers of this article to take heed and start to turn the tide in the direction whereby analysis by sex becomes the norm for all immunological and clinical studies.53 The knowledge gained would be of huge scientific and clinical importance. Funding: None. Competing interests: None declared. Ethical approval: Not required