Testosterone Influences Bladder Mechanics and Reflects Natural Sex Differences

Introduction: : The urinary bladder is a highly distensible muscular organ that repeatedly undergoes a process of filling and emptying, resulting in significant tissue deformation. Mechanics are crucial in defining the bladder’s physiological function and dysfunction. Conditions like overactive/underactive bladder and urinary incontinence lead to significant bladder tissue remodeling, affecting bladder biomechanics and potentially worsening dysfunction. Despite this, the mechanical behavior of the bladder wall is understudied in both health and disease.
Previous studies on mice have identified sex-dependent biomechanical differences in bladder function in vivo. These differences can arise from genetics, behavior, anatomy, and physiology. While human biomechanical data are lacking, research shows that the incidence of bladder dysfunction varies by sex. A better understanding of the effect of sex and sex hormones on bladder structure and function in needed. However, few studies focus on how sex affects bladder mechanics, often relying on physiological responses without fully characterizing tissue mechanics.
In this study, we use a novel mechanical testing device, the Pentaplanar Reflected Image Macroscopy (PRIM), combined with image analysis techniques to accurately quantify bladder wall stress and stretch during ex vivo pressure-volume tests in mice. Our work focuses on whether sex hormones contribute to observed biomechanical sex differences. We hypothesize that exogenous and endogenous testosterone have a similar effect on urinary bladder mechanics. To test this, we quantify bladder mechanics in female mice with and without exogenous testosterone treatment and compare the results with previously reported sex differences.

Materials and

Methods: : Bladder pressure-volume mechanical tests using the PRIM system were performed on C57BL/6 mice, divided into two groups: (1) females treated with 60-day release pellets of 5α-Dihydrotestosterone (5α-DHT) (n=5) and sham females (n=6); (2) untreated females (n=12) and males (n=15). Bladders were pressurized up to 25 mmHg for 5 cycles at a rate of 0.5 μL/s. The PRIM system output included luminal pressure and five views of the bladder, captured by a camera and four inclined mirrors. The mechanical analysis used data from the final loading cycle.
Custom MATLAB and Python scripts analyzed PRIM system images to characterize geometry during pressurization. Wall thickness was measured at 25 mmHg and estimated for other pressures using the incompressibility and spherical geometry hypotheses. Mid-wall stretch and Cauchy stress were calculated under the thin-walled pressurized spherical vessel hypothesis. Stiffness was calculated using the Holzapfel-Gasser-Ogden (HGO) strain energy function and best-fit material parameters. Bladder compliance was estimated using the change in volume over the change in pressure for the low-pressure linear region of the pressure-volume curve. A prior study (Ruetten et al., American Journal of Physiology-Renal Physiology, 2019, 317(4), F996-F1009) reported in vivo threshold pressures of 3.5 mmHg for untreated males, 5 mmHg for testosterone treated females, and 10 mmHg for untreated females (statistically higher than both the other groups). Cauchy stress, stretch, and stiffness were quantified at group-specific pressures. A t-test with Welch’s correction assessed effects of hormone treatment or biological sex on mechanical parameters at threshold pressure (p < 0.05).

Results, Conclusions, and Discussions:: Our results demonstrate that exogenous testosterone affects bladder mechanics mirroring naturally occurring sex differences. At equal luminal pressures, female + 5α-DHT mice showed larger bladder volumes compared to controls (Fig. 1a), similar to differences between untreated male and female bladders (Fig. 1b). However, as reported in Ruetten et al. (2019), micturition threshold pressure differs: control females start micturition at significantly higher pressure (10 mmHg) compared to control males (3.5 mmHg) and 5α-DHT females (5 mmHg). Thus, group-specific threshold pressures are used for comparison. Despite higher threshold pressures in females, volumes are equal between groups (Fig. 1c, d). Mechanical stretch and stress were higher in 5α-DHT-treated females compared to female controls (Fig. 1e, g), as well as in untreated male compared to females (Fig. 1f, h). Although, no stiffness difference was observed between females with and without 5α-DHT treatment (Fig. 1i), untreated female bladder stiffness was higher than in males (Fig. 1j).
To validate our analysis, we quantified ex vivo compliance, which aligned with what was observed in vivo by Ruetten et al. (2019). Specifically, treatment with 5α-DHT did not affect female bladder compliance (Fig. 1k), while female bladder compliance was lower than male (Fig. 1m), confirming the inverse relationship between compliance and stiffness.
Our findings suggest that exogenous testosterone treatment in female mice shifts urinary physiology parameters toward male characteristics. Exogenous testosterone alters not only geometrical and physiological traits, but also the mechanical properties of bladder tissue. Next, we plan to study in vivo voiding behaviors and histological characteristics to investigate whether the observed mechanical changes correspond to physiological and microstructural alterations.

Acknowledgements and/or References (Optional):: This research is supported by the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health grant number R01DK135696, and National Institute of Mental Health grant number R01 MH111604.