Sex is a neglected variable in infectious disease
Historically, we have overlooked sex as a variable in infectious disease research
[1, 2]. For example, while much of our knowledge comes from animal studies, many researchers
routinely use only male animals [3]. One of the principal reasons for this is the
argument that female animals, undergoing cyclic hormonal fluctuations, introduce additional
experimental variation [4]. Sex bias is also a major challenge in clinical studies.
In 1977, Food and Drug Administration (FDA) guidelines for human studies recommended
that women of reproductive age be excluded from early clinical trials (e.g., Phase
I) [1, 5]. While more recent efforts have resulted in greater inclusion of female
subjects [5], the lasting consequence of this recommendation is that many drug regimens
and therapeutic approaches are based solely on information gained from testing in
male subjects [5–7]. Major adverse effects experienced by female patients underline
that single-sex studies cannot predict whether and how men and women will respond
differently to a drug, vaccine, or treatment [7].
It has become increasingly clear that sex broadly influences the host immune response
[1, 2, 8]. Indeed, the influence of sexual dimorphism is likely underappreciated.
The analysis of more than 14,000 wild-type and 40,000 mutant mice revealed that approximately
10% of qualitative and more than 50% of quantitative phenotypes are influenced by
sex in wild-type mice [9]. Similarly, mutant phenotypes were impacted by sex in approximately
13% of qualitative and 17% of quantitative traits analyzed [9]. At the gene expression
level, modest but significant differences exist between male and female liver, adipose,
muscle, and brain tissue in mice [10]. In humans, as in experimental animal systems,
what we now appreciate is that men generally exhibit greater susceptibility, prevalence,
and severity of infection compared with women, which can be seen across a wide variety
of pathogens, including parasitic, fungal, bacterial, and viral infections [1, 2,
11, 12]. Exceptions to this generality, however, can be found in which susceptibility
or severity to infection, for example, is more pronounced in women. Importantly, what
drives these differences is still poorly understood. By taking a closer look at two
examples, urinary tract infection (UTI) and influenza, we can begin to appreciate
some of the many factors that likely drive these differences.
Do hormones shape susceptibility to UTI?
UTIs have a distinctive pattern in that it is women who exhibit increased susceptibility
and prevalence of infection, whereas men experience increased severity [13–15]. The
prevalence of bacteriuria, or bacteria in the urine, is approximately 10% in adult
women and 0.1% or 1/1000 men [16]. Why men experience UTIs less frequently is often
attributed to anatomical differences between men and women, including urethra length
[16]. However, several lines of evidence suggest that sex bias in UTI is driven not
only by dissimilar urethra length [16] but by sex-based variation in the levels of
specific hormones, such as testosterone or estrogen, between men and women over the
course of a lifetime. For example, UTI incidence in male infants is nearly twice that
of female infants, and in children under age 2, 40% of UTI patients are male [17,
18]. At the other end of the spectrum, the incidence of UTI in geriatric populations
(>65 years) is roughly similar between men and women (14% in women vs 11% in men)
[19]. Indeed, the sex difference in UTI is most pronounced in nongeriatric adults
[13], coinciding with the highest levels of sex hormones. Thus, UTI risk and severity
change over the lifetime of females and males, suggesting that sex hormone levels
or other sex differences contribute to differing host responses.
Supporting this idea, the elimination of estrogen in an experimental setting by ovariectomy
leads to higher bacterial burden following uropathogenic Escherichia coli infection
compared with intact mice [20]. Estrogen supplementation augments expression of the
antimicrobial gene human β-defensin 3 and strengthens urothelial junctions in vitro,
which may positively impact barrier function in the bladder, protecting against infection
[20]. Lastly, in a double-blind clinical study, topical estrogen application reduced
the incidence of recurrent UTI in postmenopausal women, with benefit attributed to
increased lactobacilli colonization and decreased vaginal pH [21]. Taken together,
these findings suggest that estrogen may play a protective role against UTI, and its
loss may make women more vulnerable to infection. Furthermore, if hormones shape susceptibility
to (uro)pathogens, hormone manipulation may alter host immunity, and—in the case of
UTI—potentially reduce incidence in women or both sexes. Additional preclinical and
clinical research is needed to address the influence of estrogen and to explore this
treatment avenue for UTI.
Hormone manipulation alters the host response to influenza
Although supplemental estrogen appears to be protective in the case of UTI, hormone
manipulation—such as contraceptive use or hormone replacement therapy—likely has a
more nuanced impact on immunity. In an influenza model, direct comparison of the two
sexes reveals that female mice exhibit greater morbidity and mortality than male mice,
potentially because of elevated levels of cytokines, such as tumor necrosis factor
(TNF)-α and C-C motif chemokine ligand 2 (CCL2), in female mice [22]. Interestingly,
a reduction in hormone levels through gonadectomy decreases mortality in female mice
and increases mortality in male mice [22]. When gonadectomized mice are supplemented
with exogenous hormone, testosterone does not impact mortality in male mice, whereas
estrogen signaling via estrogen receptor α leads to improved mortality [22]. This
estrogen-mediated protection in female mice is dependent upon alterations in cytokine
levels and the recruitment of neutrophils at later stages of infection [23]. Notably,
estrogen supplementation results in very high levels of this hormone compared with
intact, untreated female mice [22], suggesting that estrogen therapy may protect women
against influenza; however, this remains to be tested.
Specifically in female mice, progesterone treatment decreases cytokine-mediated inflammation,
induces the expansion of T helper 17 (Th17) T cells, and promotes accelerated lung
tissue healing through the expression of the tissue repair molecule amphiregulin during
primary influenza infection [24]. In addition to progesterone, the synthetic progestin
analog levonorgestrel, used in oral contraceptives, limits morbidity while reducing
serum antibody titers against a primary flu infection [25]. Despite reduced antibody
titers, animals challenged with an influenza drift variant, encoding minor changes
in sequence compared with the original virus, are protected regardless of the hormone
treatment received [25]. By contrast, challenge with a heterologous influenza virus
induces greater immune pathology and mortality, potentially mediated by decreases
in virus-specific CD8+ T cells in progesterone- or levonorgestrel-treated mice compared
with placebo-treated animals [25]. Together, these findings suggest that women using
progesterone-based contraception may experience more severe responses to subsequent
flu infection from season to season.
Finally, testosterone supplementation in aged male mice reduces clinical symptomology
and mortality following influenza infection [26]. Interestingly, testosterone does
not impact viral titer, pulmonary damage, or antibody production, leaving in question
the exact mechanisms of its action in this model [26]. Taken together, given that
the vast majority of women in the United States will use hormonal contraceptives at
some point in their lifetime [27] and that hormone replacement therapy is used in
many clinical settings in both men and women, these findings merit additional preclinical
and human studies. The findings also support that treatment options for those suffering
from infection should take into account not only the sex of the patient but their
contraceptive and hormonal status.
Nonhormonal sex-biasing differences influence host–pathogen interactions
Sex differences in infection can be mediated by more than hormonal influence [1].
The X chromosome expresses a number of immune-related genes, such as toll-like receptor
7 (TLR7) and Interleukin-1 receptor-associated kinase 1 (IRAK1), as well as a number
of immune-associated microRNAs [28]. While X inactivation, or silencing of one X chromosome,
in women would be expected to provide dosage compensation of X-linked genes, certain
regions of the X chromosome escape inactivation [28, 29]. This can lead to higher
transcription levels of specific genes, such as TLR7, leading to sex-specific responses
to viral infection [28–30]. The Y chromosome also influences immune gene expression,
regulation, and susceptibility to both noninfectious autoimmune diseases and infection
[31]. For example, the Y chromosome mediates susceptibility to cocksackievirus independently
of sex hormone expression [32]. Moving away from sex chromosomes, an analysis of eosinophil
infiltration into lymph nodes following Leishmania major infection revealed that four
autosomal loci control eosinophil numbers [33]. Of these loci, three appear to be
influenced by sex, with one of the three regulating eosinophil infiltration only in
infected male mice [33]. Additional work will be needed to determine the mechanisms
behind these phenotypes.
How can sex differences be more prominently addressed in research?
As diverse sex-based mechanisms clearly have a profound impact on disease susceptibility,
severity, and response, the challenges of considering both sexes in infectious disease
research must be addressed. The simplest step for researchers to take is reporting
the sex of the animals, cells, or cell culture models used. The journal Endocrinology
embraced this idea in 2012, specifying that the methods section of submitted manuscripts
must indicate the sex of animals used or the sex of the animal from which primary
cultures were derived [34]. Additional editors have advocated for the inclusion of
sex reporting in submitted manuscripts; however, not all have mandated that this information
is absolutely required [35]. Specifying the sex of the animal used, as well as clearly
reporting whether only one sex was used in research studies, will highlight findings
that may not be amenable to generalization to both sexes. Furthermore, some of the
perceived reasons for excluding a particular sex may not be as relevant as originally
thought. A recent meta-analysis of nearly 300 studies found that phenotypic variability
is not greater in female animals compared with male animals, even when estrous cycle
staging is not employed, dispelling the belief that female mouse studies are intrinsically
more variable [36]. Efforts such as this analysis should help allay concerns and encourage
researchers in fields that predominantly rely upon male animals (e.g., neuroscience,
physiology, pharmacology, and endocrinology [3]) to consider female animal models.
Indeed, greater efforts to include male and female animals should be made when feasible
or warranted. For example, with a single exception utilizing a surgical model of infection
[37], no studies have directly addressed the sex bias in UTI. It is the opinion of
several leaders in the field of sex-based differences that the inclusion of male and
female animals in preclinical studies will ultimately lead to reduced costs and greater
knowledge at the clinical stage [38]. Despite these obvious benefits, the inclusion
of both sexes is not always an option because of constraints such as increased associated
costs. Additionally, as research builds on published studies, findings that contradict
the literature or reveal that specific phenotypes are not maintained in the opposite
sex may face greater publishing challenges. While it will be difficult to overcome
this type of challenge, specific mechanisms, such as the National Institutes of Health
(NIH)’s administrative supplement for research on sex/gender influence, are aimed
at supporting the increased costs associated with testing in both sexes (PA-17-078).
Finally, the inclusion of women in clinical trials has increased dramatically through
the efforts of the FDA and NIH [5]. Policies such as the NIH Revitalization Act—recognizing
that the exclusion of women from early-stage clinical trials has led to a deficit
in the understanding of women’s health as well as sex-based differences—have emphasized
that sufficient numbers of women must be included in clinical research and that studies
should include specific analyses of sex-based differences [5]. The biggest challenge,
however, is that many studies are not powered for separate analyses of men and women,
which can lead to the erroneous conclusion that no differences exist between the sexes
[35]. Going forward, efforts aimed at the inclusion of both sexes in animal and human
studies, with sufficient power to analyze potential sexual dimorphism, will advance
our understanding of host–pathogen interactions and lead to targeted therapies to
safely combat infectious diseases in men and women.