The Effects of Testosterone Administration on Muscle Areas of the Trunk and Pelvic Floor in Hysterectomized Women with Low Testosterone Levels: Proof-of-Concept Study

Abstract

Objective:

To determine the effect of testosterone administration on trunk and pelvic floor muscle area in women with low testosterone levels.

Methods:

Participants were hysterectomized women with total testosterone<31ng/dl and/or free testosterone<3.5 pg/ml participating in the Testosterone Dose Response in Surgically Menopausal Women (TDSM) trial. All participants received a standardized transdermal estradiol regimen during the 12-week run-in period, and were then randomized to receive weekly intramuscular injections of placebo, or 3, 6.25, 12.5 or 25 mg testosterone enanthate for 24 weeks. Muscle areas of the trunk and pelvis were measured at baseline and end of treatment using 1.5 Tesla magnetic resonance imaging. Total and free testosterone levels were measured by LC-MS/MS and equilibrium dialysis, respectively. Testosterone effect on muscle areas were analyzed using linear regression models.

Results:

24 women who had available baseline and post-treatment MRIs were included in the analysis. Increased cross sectional areas of the paraspinal, psoas and abdominal wall muscles were seen after testosterone administration. The estimated mean change (95% confidence interval; p-value) between treatment groups was 4.07 cm² (1.26, 6.88; p=0.007) for paraspinal, 1.60 cm² (0.10, 3.09; p=0.038) for psoas major and 7.49 cm² (1.96–13.02; p=0.011) for abdominal wall muscles. Increases in psoas muscle area was significantly associated with changes in free testosterone concentrations. No significant changes in obturator internus and pelvic floor muscle areas were observed.

Conclusion:

Short-term testosterone administration in women with low testosterone levels was associated with increased trunk muscle area.

INTRODUCTION

Androgen therapy has been widely promoted in women with low serum testosterone levels for the treatment of sexual dysfunction and also for potentially improving body composition, bone mineral density, muscle performance, and physical function ¹. Testosterone levels decline progressively with age in women; and very low serum testosterone levels in older women are associated with increased risk for frailty ². Surgically menopausal women (who have low testosterone levels as a result of bilateral oophorectomy) demonstrate poorer physical performance than naturally menopausal women, regardless of estrogen therapy use ^{3, 4}, suggesting that androgens rather than estrogens may be a more important determinant of physical function in women. Androgens are known to exert direct anabolic effects on skeletal muscle in both sexes ⁵. Testosterone supplementation results in dose-dependent increases in both muscle mass and strength in men ^{6, 7}. Similarly, we have previously demonstrated that 24-weeks of testosterone administration in hysterectomized women with low testosterone levels was associated with dose and concentration-dependent gains in total lean body mass, muscle strength and physical function ⁸, further supporting the anabolic and function-promoting properties of testosterone in women.

The muscles of the trunk, which include the abdominal, paraspinal, psoas and other muscles comprising the lumbo-pelvic hip complex, are responsible for maintaining posture, balance and stability to the spinal column and therefore play an important role in physical function as well as for the prevention of falls ⁹. Trunk muscle strength, commonly referred to as ‘core strength’ or ‘core stability’, has also been shown to be positively associated with balance performance ¹⁰ as well as the ability to perform activities of daily living in older adults ⁹. Furthermore, poor trunk muscle composition (higher fat infiltration) has been demonstrated to be an important predictor for long-term functional mobility impairments (particularly loss of balance) in older adults 3-years later ¹¹, underscoring the importance of the core muscles in maintaining physical function and preventing falls. In postmenopausal women, weakness of the trunk extensor muscles have been found to be a significant predictor of increased risk for falls ¹². In addition to the stabilizing muscles of the trunk, the obturator internus muscle is also important for hip external rotation/abduction as well as hip stabilization and motion of the lower extremity and its weakness with aging could contribute to mobility limitation and falls ^{13, 14}. Despite their importance, the responsiveness of these important groups of muscles to testosterone in women is unknown.

Another important group of muscles that have relevance to aging in women are the muscles of the pelvic floor. The prevalence of pelvic floor disorders increases with advancing age, particularly in postmenopausal women ^15-17. Muscles of the pelvic floor and lower urinary tract are involved in the support of pelvic organs and micturition. In addition, voluntary contractions of these muscle groups enable control of the urethral sphincter and the urethral canal ¹⁸. The levator ani muscle and other pelvic floor muscles and structures are responsible for dynamic internal pelvic organ support in response to variations in abdominal pressure. Recent studies have shown that androgens may potentially play an important role in the pelvic floor, as the muscles in these structures, particularly the levator ani and urethral sphincter, are androgen-sensitive ^{19, 20} and may provide a therapeutic option in women with pelvic floor disorders such as urinary incontinence.

Few studies have assessed the efficacy of testosterone therapy on increasing mass of the trunk and pelvic muscles. The lack of data may be due to the inability to assess the area of these muscle groups using conventional imaging methods, such as dual-energy X-ray absorptiometry (DXA) scan. Furthermore, lean trunk mass measured by DXA scan is confounded by the abdominal parenchymal organs. Magnetic Resonance Imaging (MRI) has emerged as a useful imaging modality to quantitatively assess the trunk and pelvic muscle areas because of its soft-tissue contrast ^{21, 22}. In a randomized placebo-controlled trial of young healthy men, we recently demonstrated that exogenous testosterone administration resulted in dose-dependent increases in muscle areas of the trunk and pelvis measured by MRI, and these gains correlated with increases in serum testosterone levels ²³. However, the anabolic effects of testosterone on the trunk core and pelvic muscles have not been studied in women. Accordingly, we conducted this proof-of-concept study using MRI to evaluate the effect of testosterone administration on trunk and pelvic muscle area in hysterectomized women with low testosterone levels who had previously participated in the Testosterone Dose Response in Surgically Menopausal Women (TDSM) trial ²⁴.

METHODS

Study Design

As described previously ²⁴, the Testosterone Dose Response in Surgically Menopausal Women (TDSM) trial was a placebo-controlled, double-blind randomized trial designed to determine the dose-response effects of testosterone on a range of androgen-dependent outcomes. The trial consisted of a 12-week run-in period of transdermal estradiol administration, a 24-week treatment period, and a 16-week recovery period. The study was approved by the institutional review boards of Boston University Medical Center (BUMC) and Charles Drew University of Medicine and Science (Los Angeles, CA), and all participants gave written informed consent. The trial is registered at ClinicalTrials.gov ().

Participants

The details of eligibility and exclusion criteria have been published ²⁴. Briefly, healthy women, 21–60 years of age who had undergone hysterectomy (with or without partial or total oophorectomy) and with low serum testosterone levels (total testosterone < 31ng/dl or free testosterone < 3.5pg/ml (below the median for healthy young women ²⁵) were eligible to enroll in the study. Women with major psychiatric illness, poorly controlled diabetes mellitus (HbA1c >8.5%), uncontrolled hypertension, severe obesity (BMI >40 kg/m²), abnormal liver function, history of breast, ovarian, endometrial or cervical cancer, hyperandrogenic disorders, cardiac disease, thromboembolic disorders, or were receiving glucocorticoids, androgens, spironolactone and GnRH agonists were excluded.

Randomization and Study Interventions

All eligible women were administered a regimen of transdermal estradiol (E2) patch applied twice a week and designed to achieve nominal delivery of 50-ug estradiol daily (Alora, Watson Pharmaceuticals, Morristown, NJ, USA) for a 12-week run-in phase. After run-in, the participants were randomized in a double-blinded fashion to one of 5 groups to receive weekly IM injections of placebo, 3, 6.25, 12.5 or 25 mg testosterone enanthate (ENDO Pharmaceuticals, Malvern, PA, USA) for 24 weeks.

Hormone Assays

Serum total testosterone levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with sensitivity of 2 ng/dl ²⁶. The interassay coefficient of variation was 15.8%, at 12.0 ng/dL, 10.6%, at 23.5 ng/dL, 7.9%, at 48.6 ng/dL, 7.7% at 241 ng/dL, 4.4% at 532 ng/dL, and 3.3% at 1016 ng/dL respectively. Free testosterone was measured using equilibrium dialysis with an interassay coefficient of variation of 12.3% with sensitivity of 0.3 pg/ml ^{25, 27}. Sex hormone binding globulin levels were measured using an immunofluorometric assay with a sensitivity of 0.5 nmol/L ⁶.

Magnetic resonance imaging (MRI)

All women underwent 1.5 Tesla magnetic resonance imaging (MRI; Philips Medical Systems of North America) of the abdomen and pelvis, using a respiratory-triggered mixed fast spin-echo-pulse sequence at baseline and the 20^th week of treatment, generating parametric T1 maps. The image matrix size was 256×256 or 320×320, axial slice thickness: 7 mm at L3 and 6–8 mm in the pelvis; interslice distance: 2 mm. The changes in the trunk was analyzed at echo time (TE) 6.2–6.5/9.5 and repetition time (TR) 1500–5000; pelvic muscle area was analyzed at TE 6.4/9.5/11.1 and TR 3360–4900/900/5900–6400. Settings varied between participants but only marginally within participants. A single experienced operator (JT), blinded to treatment assignment, located and manually traced the relevant muscles separately and their areas were calculated in an automated fashion using OsiriX version 7.5.1 (Pixmeo, Bernex, Geneva, Switzerland). The inter-reader and intra-reader reproducibility of manual analysis of body composition using MRI have previously shown to be reliable ^{28, 29}. The image analysis of the trunk muscles was done in a single section at the third lumbar vertebrae (L₃) ^{28, 30-33}.

Outcome measurements

Trunk Muscles:

• Psoas Muscle: the areas of both psoas major muscles;

• Paraspinal Muscle: the total area of the lumbar paraspinal muscles (erector spinae and quadratus lumborum);

• Total Abdominal Muscle: the sum of the areas of psoas major muscle, paraspinal muscles and the anterior abdominal wall muscles (transverse abdominis, internal and external obliques, and rectus abdominis muscles) at the level of L₃. (Figure 1A). Some images were affected by motion artifact, presumably due to breathing during the scan, to the level that accurate measurements were deemed not possible and thus excluded from the analysis.

Figure 1. T1-weighted Magnetic Resonance Images of areas Measured.

A) Muscle areas at 3^rd lumbar vertebrae, abdominal wall (AW), psoas major (PM), paraspinal muscles (PS), B) The obturator internus muscles, C) The pelvic floor muscles.

Obturator Internus Muscle

Origin: the membrane of the foramen obturatum. Insertion: the major trochanter of the femur. Function: stabilization of the hip joint, assisting in outwards rotation of the thigh. The obturator internus muscle area was measured at the level with the largest combined trans-axial muscle widths (Figure 1B).

Pelvic Floor Muscle

Pelvic floor muscles, including the levator ani muscle, are situated in the lower part of the pelvis. Function: important for urinary and fecal continence; support of abdominal and pelvic organs. The pelvic floor muscles were measured using an axial slice at the level of the distal part of the symphysis pubis. The rectum, vagina and the urethra were excluded from measurement. (Figure 1C). In some of the images, the MRI settings and resolution were not optimized and did not allow us to fully delineate between different layers of tissue (i.e. pelvic floor, anal canal, vagina and urethra); thus, the pelvic floor muscle area could not be accurately measured in these participants and were therefore excluded from the analysis.

Statistical Analysis

Analyses were performed on the subset of women (n=24) who underwent MRI at baseline and 24 weeks. Descriptive statistics for the placebo and testosterone groups (all testosterone doses combined) were presented as means and standard deviations. Model assumptions and distribution of outcomes were investigated graphically. Outcomes were expressed as change in muscle area from baseline to 24-week follow-up. Crude association of muscle measurements with on-treatment testosterone levels was explored using linear regression models. Magnitude of this relationship was assessed by regression slopes and 95% confidence limits along with R-squared and corresponding p-values. Testosterone effect was assessed by linear regression models with treatment factor adjusted for baseline measurements and BMI. Due to the small sample size, the muscle area measurements for all individuals randomized to any testosterone dose were combined and compared to placebo to increase statistical power for this proof-of-concept analysis. All statistical tests were two-sided with alpha level 0.05. Analyses were performed using SAS v.9.4 (SAS Institute, Cary, NC) and R software version 2.15.1.

RESULTS

Participants

Of the 850 women who underwent telephone screening, 218 met eligibility criteria, 85 entered the estrogen run-in-period, 71 were randomized, and 24 who had baseline and end-of-treatment MRI data constituted the analytic sample (placebo (n=7), all testosterone dose groups combined (n=17). The number of individuals within each dose group of testosterone enanthate were as follows: 3 mg (n=5), 6.25 mg (n=4), 12.5 mg (n=4) or 25 mg (n=4). Due to small sample size, individuals randomized to any testosterone dose were combined into one group and compared to individuals randomized to the placebo group for this analysis.

Baseline characteristics of the placebo group and the testosterone-treated group (participants randomized to any of the 4 testosterone doses) are displayed in Table 1. Mean age of women enrolled in the study was 51.8 years (range 41–60) with an average BMI of 30.1 kg/m².

Table 1:

Baseline Characteristics of Participants Treated with Testosterone versus Placebo

Variable	Testosterone Group (N=17)	Placebo Group (N=7)	Total (N=24)
Age, years	50.8 ± 5.2	54.4 ± 5.5	51.8 ± 5.4
Body Mass Index, kg/m2	28.5 ± 6.2	34.0 ± 1.6	30.1 ± 5.8
Prior Pregnancies, number	2.0 ± 1.3	1.4 ± 1.1	1.9 ± 1.3
Total Testosterone, ng/dL	14.8 ± 11.3	12.5 ± 6.4	14.1 ± 10.0
Free Testosterone, pg/mL	2.5 ± 2.1	2.0 ± 0.83	2.3 ± 1.8
Sex Hormone Binding Globulin, nmol/L	55.0 ± 26.1	75.1 ± 31.5	61.1 ± 28.7
Psoas Muscle Area, cm2	11.7 ± 2.6	13.2 ± 2.1	12.1 ± 2.5
Paraspinal Muscle Area, cm2	43.2 ± 6.2	43.7 ± 8.4	43.4 ± 6.7
Obturator Internus Area, cm2	17.3 ± 2.2	15.9 ± 2.5	16.8 ± 2.3
Abdominal Muscle Area, cm2	56.5 ± 12.7	57.5 ± 10.4	56.8 ± 11.8
Pelvic Floor Muscle Area, cm2	8.7 ± 3.2	10.1 ± 2.9	9.1 ± 3.1

Data represent mean ± SD; Testosterone Group is comprised of participants randomized to any of the 4 different testosterone dose groups.

Hormone Levels

Baseline mean total and free testosterone concentrations were 14.1 ng/dl and 2.3 pg/ml, respectively, well below the range for healthy, menstruating women ²⁴. Serum nadir total and free testosterone levels, measured during week 24, one week after the previous injection, increased from baseline in a dose-dependent fashion, resulting in levels that were in physiologic to supraphysiologic range. Mean on-treatment nadir total testosterone concentrations were 13.9, 96.0, 172.1, 179.6 and 252.1 ng/dl, and free testosterone concentrations were 2.3, 17.6, 31.1, 30.6 and 49.3 pg/ml at the 0, 3, 6.25, 12.5 and 25-mg doses, respectively. Fifteen out of seventeen participants (88.2%) in the testosterone-treated group had supraphysiological levels of testosterone (total testosterone >60 ng/dl post-intervention).

Changes in trunk muscles

Testosterone administration (all doses combined) was associated with significant increase in paraspinal cross-sectional area compared to placebo (estimated mean difference between treatment groups; +4.07 cm², p=0.007; Table 2A, Figure 2). The change in paraspinal muscle area were not related to increases in serum total or free testosterone concentrations (Table 3, Figure 3). The change in paraspinal muscle area ranged from an increase of 4.6% in the lowest testosterone dose group to an increase of 9.7% in the highest dose group.

Table 2A.

Estimated mean difference of trunk and pelvic floor muscle areas between treatment groups

Treatment Group	Placebo		Testosterone		Estimated mean difference between treatment groups, mean (95% CI)	P-value	Sample Size, N
	Baseline	Post Treatment	Baseline	Post Treatment
Psoas Muscle Area, cm2	13.22 ± 2.14	13.31 ± 2.53	11.66 ± 2.59	13.09 ± 2.96	1.60 (0.10, 3.09)	0.04	24
Paraspinal Muscle Area, cm2	43.65 ± 8.37	42.49 ± 8.89	43.24 ± 6.20	45.51 ± 6.42	4.07 (1.26, 6.88)	0.01	24
Obturator Internus Area, cm2	15.90 ± 2.45	15.60 ± 1.42	17.13 ± 2.16	17.84 ± 2.39	0.82 (−0.94, 2.58)	0.34	24
Abdominal Wall Muscle Area, cm2	57.48 ± 10.44	57.71 ± 8.33	56.45 ± 12.68	59.31 ± 12.19	7.49 (1.96, 13.03)	0.01	23
Pelvic Floor Muscle Area, cm2	10.14 ± 2.93	8.77 ± 1.66	8.81 ± 3.09	8.60 ± 2.61	1.91 (−0.29, 4.11)	0.08	18

Values are expressed as mean ± std for each treatment group, mean and 95% confidence intervals (CI) for group difference. P-values and estimates are extracted from linear regression model adjusted for baseline muscle area and body mass index. The estimated mean difference between treatment groups is the difference between post-treatment change from baseline in muscle area in the testosterone group and post-treatment change from baseline in muscle area in the placebo group

Figure 2. Change in Trunk and Pelvic Muscle Area by Treatment Group.

Boxplots present the mean and 95% confidence interval of 24 weeks changes from baseline in trunk and pelvic muscle areas by treatment group. P-values are extracted from linear regression models adjusted for baseline muscle area and BMI.

Table 3.

Relationship between changes in trunk and pelvic floor muscle areas with changes in serum testosterone concentrations in participants randomized to testosterone intervention

Variable	N	β estimate (95% CI)	p-value	R2
Psoas Muscle, cm2
Total Testosterone, 100 ng/dl	17	0.60 (−0.11, 1.31)	0.094	0.176
Free Testosterone, 10 pg/ml	17	0.37 (0.01, 0.73)	0.043	0.246
Paraspinal Muscles, cm2
Total Testosterone, 100 ng/dl	17	0.57 (−0.73, 1.87)	0.364	0.055
Free Testosterone, 10 pg/ml	17	0.31 (−0.37, 0.99)	0.345	0.060
Obturator Internus, cm2
Total Testosterone, 100 ng/dl	17	−0.38 (−1.31, 0.55)	0.398	0.048
Free Testosterone, 10 pg/ml	17	−0.14 (−0.63, 0.35)	0.552	0.024
Abdominal Wall Muscles, cm2
Total Testosterone, 100 ng/dl	16	−0.16 (−3.43, 3.12)	0.920	0.001
Free Testosterone, 10 pg/ml	16	0.11 (−1.62, 1.84)	0.891	0.001
Pelvic Floor Muscles, cm2
Total Testosterone, 100 ng/dl	13	−0.64 (−1.48, 0.19)	0.117	0.208
Free Testosterone, 10 pg/ml	13	−0.19 (−0.67, 0.29)	0.402	0.065

Values are expressed as β parameter estimates and 95% confidence intervals (CI) of muscle area change per 100 ng/dl increase in total testosterone and per 10 pg/ml increase in free testosterone levels. P-values and R² are extracted from simple linear regression models.

Figure 3. Change in trunk muscle areas with change in serum testosterone levels.

Scatter diagram of serum testosterone change on trunk muscle areas after 24 weeks in participants treated with testosterone. P-values and R²represent association between trunk muscle areas and serum testosterone calculated from simple linear regression models. T, testosterone.

The testosterone-treated group (all doses combined) showed a significant increase in psoas cross-sectional area compared to placebo (estimated mean difference between treatment groups; +1.59 cm², p=0.038; Table 2A, Figure 2). Serum free testosterone levels were positively associated with increases in psoas muscle area (R²=0.246, p= 0.043) (Table 3, Figure 3). The change in psoas muscle area ranged from an increase of 11.7% in the lowest testosterone dose group to an increase of 18.9% in the highest dose group (25-mg/wk.).

Testosterone administration (all doses combined) resulted in a significant increase in abdominal muscle area compared to placebo (estimated mean difference between treatment groups; +7.49 cm², p=0.011; Table 2A, Figure 2). The increase in abdominal wall muscle area was not associated with change in serum testosterone levels.

Changes in pelvic muscles

There were no significant changes in obturator internus and pelvic floor muscle areas when compared to placebo. Changes in obturator internus and pelvic floor muscles were not related to increases in serum testosterone concentrations.

Further subanalyses comparing women in the testosterone arm who had on-treatment supraphysiological total testosterone levels (>60 ng/dl) with placebo showed similar results. These results are shown in Table 2B.

Table 2B.

Estimated mean difference of trunk and pelvic floor muscle areas between placebo group and participants in the testosterone group with supraphysiologic testosterone Levels.

Treatment Group	Placebo		Testosterone		Estimated mean difference between treatment groups, mean (95% CI)	P- value*	Sample Size, N
	Baseline	Post Treatment	Baseline	Post Treatment
Psoas Muscle Area, cm2	13.22 ± 2.14	13.31 ± 2.53	11.42 ± 2.54	12.76 ± 3.00	1.43 (−0.02, 2.89)	0.05	22
Paraspinal Muscles, cm2	43.65 ± 8.37	42.49 ± 8.89	43.60 ± 6.02	45.76 ± 6.26	3.94 (0.92, 6.96)	0.013	22
Obturator Internus, cm2	15.90 ± 2.45	15.60 ± 1.42	17.37 ± 2.27	18.26 ± 2.40	0.94 (−0.95, 2.83)	0.31	22
Abdominal Wall Muscles, cm2	57.48 ± 10.44	57.71 ± 8.33	56.51 ± 12.21	59.20 ± 11.56	7.27 (1.44, 13.10)	0.018	21
Pelvic Floor Muscles, cm2	10.14 ± 2.93	8.77 ± 1.66	8.54 ± 3.13	8.63 ± 2.72	1.90 (−0.40, 4.19)	0.10	17

Values are expressed as mean ± std for each treatment group, mean and 95% confidence intervals (CI) for group difference. P-values and estimates are extracted from linear regression model adjusted for baseline muscle area and body mass index. The estimated mean difference between treatment groups is the difference between post-treatment change from baseline in muscle area in the testosterone group and post-treatment change from baseline in muscle area in the placebo group

DISCUSSION

In our trial of hysterectomized women with low testosterone levels, short-term testosterone administration for 6 months was associated with increases in cross sectional area of paraspinal, abdominal wall and psoas muscles. Of the trunk stabilizing muscles evaluated in our study, the psoas muscle specifically correlated with on-treatment serum free testosterone levels. No significant response to testosterone was demonstrated in the obturator internus muscle nor in the pelvic floor muscles. We have recently showed that exogenous testosterone administration was associated with dose and concentration-dependent increases in the trunk muscles of young healthy men ²³. Similar to men, our findings suggest that these trunk stabilizing muscles in women are also responsive to testosterone administration and can be potential therapeutic targets in frail older women who are at high risk for falls.

The muscles of the trunk play a critical role in influencing balance and mobility status by providing dynamic stability to the vertebral column ^{34, 35}. Thus, atrophy of these muscles can increase susceptibility to mobility limitation and falls ³⁴. A previous longitudinal study of men who have been treated for rectal cancer with surgery and radiotherapy demonstrated that decline in endogenous serum testosterone levels associated with loss of psoas muscle area ³⁶. Similarly, patients with hypercortisolism also demonstrated significant atrophy of the psoas major muscle ³⁷. These studies provide evidence that the trunk muscles are influenced by both anabolic and catabolic endogenous hormones and may provide a therapeutic target for patients with sarcopenia and physical function impairments.

Prior studies investigating the exogenous effects of testosterone on muscle strength and physical function outcomes in older adults have primarily focused on peripheral musculature (i.e. appendicular lean mass and muscle strength)^{6, 38}; with only few studies that have explored testosterone effects on muscles of the trunk ^{23, 39}. Furthermore, the data available on the anabolic effects of testosterone on these specific muscles are limited to men and have not been previously studied in women. Our study shows that exogenous testosterone administration to hysterectomized women with androgen deficiency results in significant increases in core muscle area assessed by MRI. Furthermore, increase particularly in the psoas major muscle is associated with changes in free serum testosterone levels. One small trial conducted in hypogonadal men (age 18–74 years) reported increases in area of the paraspinal muscles assessed with computed tomography ³⁹. Similarly, in a larger trial in young healthy men (age 18–50), we recently showed that testosterone dose-dependently increased several muscle areas of the trunk, which included the psoas, total abdominal and paraspinal muscles assessed by MRI; and the increase in these muscle areas was significantly associated with changes in on-treatment total and free testosterone levels ²³. Consistent with these studies in men, our findings also provide evidence that the muscles of the trunk are responsive to testosterone in women which has not been previously reported.

Older women are at increased risk of urinary incontinence and pelvic floor disorders, and these conditions are significantly associated with poor quality of life ^40-43. Recent studies have shown that androgens may potentially play an important role in the pelvic floor and lower urinary tract, as the muscles in these structures, particularly the levator ani and urethral sphincter, are androgen-sensitive ^{19, 20}. These pelvic floor muscles also express large numbers of androgen receptors in both animals and humans ⁴⁴. This is further supported in observational studies in hyperandrogenic women with polycystic ovary syndrome, who demonstrate both stronger muscle tone and stronger voluntary muscle contraction of the pelvic floor muscles compared to healthy controls ⁴⁵. Given that androgen receptors have been shown to be expressed throughout the pelvic floor and lower urinary tract, it is conceivable that testosterone administration may result in increases in the pelvic floor muscles. In our study, we did not find significant changes in pelvic floor muscle area with 24-weeks of exogenous testosterone administration. However, this lack of effect may have been due to small sample size. Thus, the anabolic effects of testosterone on pelvic floor muscles needs further study in larger and longer trials, and particularly in women with pelvic floor disorders who are experiencing urinary incontinence and/or organ prolapse.

Our study has notable strengths and some limitations. The trial had many features of a good trial design: concealed randomization, placebo-control, blinding and oversight by an independent DSMB. Total and free testosterone levels were measured using LC-MS/MS and equilibrium dialysis respectively; both widely considered the reference methods with the highest sensitivity and specificity. Testosterone injections were effective in raising testosterone concentrations in a dose-dependent fashion over a wide range. Image analysis, using MRI or computed tomography, is considered to be the state-of-the-art method in the assessment of size in the investigated muscles. In using a single expert analyst (J.T.), the inter-observer variability was eliminated. By performing manual tracking and tracing, and having access to several stacks of axial slices, the best slice for each given muscle could be selected for analysis allowing exact tracking of muscles ^{33, 46}. Manual tracing, as conducted in this study, can mitigate for potential image artifacts. The limitation of this study is that measurement of trunk and pelvic muscle areas was not the primary outcome of the trial, and the trial was not powered to detect a difference in changes in these muscle parameters. Our intention was not to measure functional outcomes associated with muscles of the trunk. Further study will be necessary to determine if the muscle cross-sectional area changes we observed are associated with functional improvements such as balance, stability, and urinary continence. Furthermore, the sample size was relatively small as the number of available MRIs was limited to a very small subset across the 5 dose groups. The main goal of this proof-of-concept study was to determine whether the mass of the trunk and pelvic floor muscles can be increased by administration of testosterone. As a result, we combined muscle area measurements for all individuals randomized to any testosterone dose to yield two treatment groups in order to increase the statistical power of our analysis and generate preliminary estimates of effect size: testosterone (all doses combined) versus placebo. In this study, the vast majority of women in the testosterone arm achieved supraphysiological testosterone levels (88.2%) regardless of testosterone dose. Based on our prior studies, the significant gains in sexual function and total lean body mass were achieved in women receiving the highest 25-mg testosterone dose group²⁴ (when compared to placebo) where highly supraphysiological testosterone levels were achieved; in addition, we demonstrated significant overall dose effects. In this substudy, we still found significant increases in trunk muscle areas when restricting our analyses to women achieving supraphysiological testosterone levels regardless of testosterone dose. Our findings demonstrate that the increase in trunk muscle areas are occurring at supraphysiological testosterone levels particularly in the highest testosterone dose group. There may be an overall dose response effect that could not be seen due to combining the testosterone dose groups as we were underpowered to perform a global test. Thus, our findings lay the groundwork in support for future larger randomized efficacy trials investigating dose-dependent effects of androgens on trunk and pelvic muscles in women.

Conclusion

We found that short-term testosterone administration for 24-weeks in hysterectomized women with low testosterone levels was associated with increases in cross-sectional area of the muscles that stabilize the trunk but not in the muscles of the pelvic floor. Based on these findings, trunk muscles could serve as a potential therapeutic target for intervention with testosterone in frail older women with physical dysfunction who are at subsequent risk for falls. The efficacy of testosterone therapy on trunk stabilizing and pelvic floor muscles in women needs further investigation in long-term, adequately powered trials.