Sex-specific components of frailty in C57BL/6 mice

Introduction

Aging is characterized by a gradual decline in various health parameters across multiple biochemical, physiological and behavioral systems [1]. With dysfunction of these systems, quality of life is impaired (i.e., reduced healthspan), which ultimately leads to death. Interestingly, a subset of aging individuals appears to lack resilience within these systems, a condition termed frailty. Frailty has broadly been defined as an age-associated biological syndrome characterized by an exaggerated vulnerability to adverse global health outcomes, a reduced capacity to react to stressors and an overall loss in physiological function [2,3]. Consequently, frail individuals are at a greater risk of falls, dependency, disability, institutionalization, hospitalization and mortality [4,5]. Studying frailty has therefore become a pivotal area of research.

Questions in the field of frailty have largely gone unanswered due to the inherent limitations of clinical studies, including biological, ethical and logistical complications of working with older individuals [6,7]. The recent development of assessment tools for frailty in mice reverse-translated from human frailty models has made it possible to bypass many of these aforementioned limitations [8]. For instance, Howlett and colleagues [7] established a mouse frailty index based on accumulated deficits derived from Rockwood et al. [9,10], that assessed 31 invasive and noninvasive variables (i.e., activity levels, hemodynamic measures, body composition and basic metabolic status), which was later simplified to only include readily apparent noninvasive signs of clinical deterioration [11]. Similarly, Thompson and colleagues [8] developed a frailty phenotype in aged mice that was similar to the clinical criteria used by Fried et al. [5], which included grip strength, walking speed, physical activity and endurance. They recently modified the phenotype to also include body weight [12,13]. The development of rodent frailty assessment tools represents an important step forward in how we understand frailty, and interventions that may delay or prevent its progression.

Recent efforts to understand frailty have extended beyond this biological syndrome solely being age-dependent, but rather age- and sex-dependent. Many age-related biochemical, physiological and behavioral changes are already known to be sex-specific [14–17], which gives credence to the premise that sex may also be an important variable associated with frailty status. In fact, women have higher frailty index scores than men at all ages but paradoxically have a lower mortality risk, a concept termed the sex-frailty paradox [18,19]. A phenomenon also known more broadly as the male-female health-survival paradox; women have poorer health than men, characterized by greater burden of chronic disease, more disability and worse self-rated health, but live longer [20,21]. These data suggest each sex may have different physiological reserves, and that the link between health status and mortality is not necessarily fixed in humans. To date, very little is known regarding sex differences in frail mice or if the sex-frailty paradox extends beyond humans. The purposes of this study were therefore to characterize sex differences in frail C57BL/6 mice across the lifespan using a validated frailty phenotype, and assess how closely it relates to the human sex-frailty paradox.

Results

A Kaplan-Meier survival analysis revealed male mice lived 13% longer than female mice (mean lifespan: 31.8±0.7 vs. 28.1±0.6 months; p<0.01) (Figure 1).

To assess sex differences in the prevalence of frailty across the lifespan, cut-off values determined at 23 and 20 months for males and females (Table 1, also see ‘Frailty criteria’ in the METHODS), respectively, were used to categorize mouse frailty from 17 months until death [12,13]. There were more frail female mice at 26 months than frail male mice (44.4 vs. 73.7%; p=0.05) (Figure 2A), however when normalized to the mean lifespan for each sex, no differences were detected (p≥0.10) (Figure 2B).

Frail mice were then assessed using a Kaplan-Meier survival analysis to determine sex differences on mortality risk (Figure 3A & B). Frail female mice (n=10) had a mean survival time of 26.7±0.8 months, which was not different (p=0.12) from the mean survival time of 28.7±0.9 months observed in frail male mice (n=9) (Figure 3A). There was also no difference between mortality rate of frail male and female mice when evaluated by survival following the frailty assessment (p=0.78) (Figure 3B). See ‘Frailty criteria’ in the METHODS for details on the frailty assessment components.

To identify if non-frail and frail mice presented anthropometrical and/or functional sex differences early in life, male and female mice were compared at the time of the frailty assessment and three months prior. Frail mice, regardless of sex, were heavier (p≤0.05) (Figure 4A and 5A) and possessed more body fat (p≤0.05) (Figure 4B and 5B) than non-frail mice. However, non-frail and frail female mice had 53.1-62.2% more body fat compared to non-frail and frail male mice (p≤0.01) (Figure 4B and 5B).

As with body weight and body fat percentage, time to fatigue (i.e., endurance, Figure 4E) at the frailty assessment and time on the rotarod (i.e., walking speed, Figure 5C) in the months prior were less (p≤0.05) for frail mice compared to non-frail mice. Frail females were weaker than non-frail females (p≤0.05), but both were stronger than males at the time of the frailty assessment (p≤0.03) (Figure 4D). In the months prior, non-frail males had better endurance scores compared to non-frail females (p=0.01) (Figure 5E). Physical activity measured by voluntary wheel run distance was 65.2-65.7% greater in non-frail females compared to non-frail males, and 127.1-170.8% greater than that of frail females (p≤0.01) (Figure 4F and 5F).

Discussion

The purposes of this study were to characterize sex differences of frail mice across the lifespan using a validated frailty phenotype, and assess how closely it relates to the human sex-frailty paradox. Our results generated three primary findings. First, the prevalence of frailty was greater in female mice but was age-dependent, and if normalized to mean lifespan, no sex differences remained. Second, frail mice, regardless of sex, died at a similar rate after the frailty assessment and had comparable mean lifespans. Lastly, anthropometrical and functional sex differences impact the selection of frailty criteria and determination of cut-off values. These results do not fully support the sex-frailty paradox observed in humans, but do suggest that regardless of sex, frail C57BL/6 mice are anthropometrically and functionally dysfunctional, and die early in life [12,13].

Women experience greater levels of frailty, which is associated with poor health and an increased risk of death, but paradoxically live longer than men [18,19]. This phenomenon has been termed the male-female health-survival paradox [20,21], also coined the sex-frailty paradox [18,22]. To date, it is currently unknown if this paradox extends to common rodent models (e.g., C57BL/6 mice) used to study frailty and aging. For comparison between human and rodents, we have outlined the sex-frailty paradox into two components: 1) the prevalence of frailty and 2) the mortality risk of those identified as frail.

The first component of the sex-frailty paradox in humans is that frailty scores are greater in women. In the laboratory, this component has not been fully established in mice. In fact, some report no sex differences [7,23] while others report greater frailty scores in female C57BL/6 mice [11], and more recently in female NIH Swiss mice [24]. Our data partially support the latter findings that female mice exhibit greater levels of frailty, though it was age-dependent and only transient. Using a longitudinal lifespan study design with a phenotypic frailty assessment tool, we observed that at 26 months of age the prevalence of frailty was 66.0% greater in females than males (73.7 vs. 44.4%), however no differences were detected at any other age (Figure 2A). Furthermore, the prevalence of frailty in males increased to a similar extent from 26 to 29 months as that seen in females from 23 to 26 months. Aging female NIH Swiss mice also experience a sharp increase in frailty scores beyond that of males when assessed using a physiological frailty index [24]. It is possible that these observations [present study, 24] are linked to hormones, immune function or regional distribution of body fat, factors associated with sex differences in response to life-extending genetic or pharmacological interventions [25]. Together, these data partially support that the prevalence of frailty is greater in females but is likely age-dependent, as all mice (regardless of sex) eventually become frail.

The observation that female mice have greater frailty scores or prevalence of frailty is not a certainty and with further interpretation, can be challenged. Indeed, in the present study male mice lived 13% longer than female mice (Figure 1). Several other groups have shown C57BL/6 male mice live longer than female mice, though there is a fair amount of variability [26,27]. Austad and Fischer [28] recently reviewed 29 studies on the C57BL/6 mouse strain and found that males lived longer in 18 studies and females lived longer in 11 studies. Therefore, it is critical researchers consider sex differences in lifespan and make the proper adjustments to their frailty scores and cut-off values. In the present study, frailty cut-off values were selected at different time-points based on the 3.7-month variance in mean lifespan between sex, with frailty determination occurring at 23 and 20 months for male and female mice, respectively (Table 1). Taking this rationale a step further, we normalized the prevalence of frailty in male and female mice to the mean lifespan for each sex, which therefore takes into account the three month offset previously mentioned (i.e., 23 vs. 20 months). Using this approach, the sex difference observed in the prevalence of frailty was eliminated, indicating that the prevalence of frailty was similar between male and female C57BL/6 mice (Figure 2B). These results and their subsequent interpretations suggest that sex differences in lifespan be taken into account when comparing male and female rodents.

The second component of the sex-frailty paradox is that women live longer than men despite having higher frailty scores [18,19]. Our data does indicate that at one time-point (without normalization to the mean lifespan), the prevalence of frailty was greater in females (Figure 2A). However, when assessing mortality risk of frail mice across sex, we did not observe that frail female mice lived any longer. In fact, regardless of sex, frail mice died at a similar rate following the frailty assessment at 23 and 20 months for male and female mice, respectively (Figure 3A & 3B). Approximately 50% of the frail male and female mice died within six months after being identified as frail (Figure 3B). Our data suggest frail male and female C57BL/6 mice die at a similar rate when frailty cut-off values are adjusted to reflect variability in sex and lifespan. Overall, in our cohort of aging C57BL/6 mice, it appears frail females do not paradoxically live longer than frail males; and therefore, does not support the second component of the sex-frailty paradox observed in humans.

After determining the prevalence of frailty and overall mortality risk did not appear to have prominent sex differences, we turned to examining sex-specific characteristics used in the frailty assessment tool. Kane et al. [29] recently articulated the concept that, “there are different ways to become frail and each of them is valid for the person or mouse who becomes frail in that way,” a concept we explored further and assessed whether it was also sex-specific. Our results indicate that although some frailty markers were similar between sex, others appeared to be dependent on being male or female. For instance, non-frail female mice were more physically active than non-frail males, a common sex difference observed in mice [30,31]. It is therefore important to acknowledge that some sex differences will be present when determining frailty criteria and cut-off values. The observation that non-frail females ran ~65% further on the voluntary wheels than non-frail males is a prime example of a functional sex difference (Figure 4F & 5F), and solid justification that frailty cut-off values cannot simply be applied across sex. Fried’s human phenotype [5] acknowledged this by using cut-off percentiles according to gender and Antoch et al. [24] alluded to this in mice, by reporting parameters of their physiological frailty assessment, which only showed partial overlap between males and females. We suggest that sex differences be considered when assessing frailty, and emphasize that there are different ways to become frail – one of which may be sex.

In closing, using a longitudinal lifespan study design we characterized sex differences in frail C57BL/6 mice via a validated frailty phenotype, and compared our findings to that of the sex-frailty paradox observed in humans. Our results suggest phenotypic sex differences exist in C57BL/6 mice and that males live longer than females. Researchers should therefore be cognizant of the anthropometrical, functional and lifespan differences between males and females, and adjust frailty criteria based on sex and mean lifespan – as done in the present study. Despite these sex differences, the prevalence of frailty was generally the same between sexes, with the exception of a single age when frailty was greater in females. Moreover, after a mouse was identified as frail, male and female mice died at a similar rate. Together, these results (i.e., in C57BL/6 mice) do not fully support the sex-frailty paradox observed in humans. In closing, these data indicate that sex differences exist in aging C57BL/6 mice and if the frailty criteria are adjusted accordingly, the prevalence of frailty increases across age with frail mice dying early in life [12,13], regardless of sex.

Methods