- Email: [email protected]
Age effects on urethral striated muscle I. changes in number and diameter of striated muscle fibers in the ventral urethra
Age effects on urethral striated muscle I. Changes in number and diameter of striated muscle fibers in the ventral urethra Daniele Perucchini, MD, John O. L. DeLancey, MD, James A. Ashton-Miller, Msc, PhD, Ursula Peschers, MD, and Tripti Kataria, MD Zurich, Switzerland, and Ann Arbor, Mich OBJECTIVE: This study was undertaken to test the null hypothesis that the number of striated muscle fibers in the ventral wall of the female urethra remains constant with increasing age. STUDY DESIGN: The urethra and surrounding tissues from 25 female cadavers, mean age 52 years (±SD 18, range 15-80 years), were selected for this study. Each specimen was divided along the midsagittal plane, and a Masson trichrome histologic section was prepared. A systematic count of striated muscle fibers in the ventral wall was then obtained at each decile of urethral length. RESULTS: A decrease in the total number of fibers within the sampled area was found with increasing age. The mean of the total fibers across all urethrae was 17,423 (±SD 9,624, range 4,788-35,867). Over the life span, an average of 364 fibers (2%) were lost per year (95% CI 197-531; P < .001). Mean fiber density was 671 (± SD 296, range 228-1374) fibers/mm2 and decreased by 13 fibers/mm2 per year (95% CI 8-17; P < .001). The mean lesser fiber diameter was 24 µm and did not change significantly with age (P = .3). CONCLUSIONS: The number and density of urethral striated muscle fibers decline with age. (Am J Obstet Gynecol 2002;186:351-5.)
Loss of urethral support and decreased urethral function can result in stress urinary incontinence in women.1-3 Although several authors have described the anatomic defects responsible for urethral hypermobility,4-6 the anatomic changes responsible for decreased urethral closure pressure have received less attention.7,8 In the female urethra, closure pressure is known to be developed principally by contraction of the smooth and striated muscle. Because a decrease in muscle cells might explain the observed closure pressure decrease with age,2,9 we hypothesized that a study of striated muscle histomorphometry might give useful insights into the underlying mechanisms of urethral dysfunction. All elements of the striated urogenital sphincter muscle are found in the ventral urethral wall. The sphincter urethrae, the urethrovaginal sphincter, and the compressor urethrae are all present in the ventral wall, whereas the dorsal wall contains only the sphincter urethrae because the urethrovaginal sphincter and the compressor urethrae diverge from the urethral wall to From the Department of Obstetrics and Gynecology, University of Michigan, and the Department Frauenheilkunde, Universitätspital Zurich. Supported by a grant from the Swiss National Science Foundation and a National Institutes of Health Grant R01 DK 47516-03. Received for publication February 15, 2001; revised June 6, 2001; accepted October 2, 2001. Reprint requests: Daniele Perucchini, MD, Departement Frauenheilkunde, Universitatsspital Zurich, Frauenklinikstrasse 10, CH-8091 Zurich, Switzerland. E-mail: [email protected] Copyright 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/121089 doi:10.1067/mob.2002.121089
pass laterally toward the ischiopubic ramus and the vaginal wall (Fig 1).10 This study focuses on the striated urogenital muscle layer in the ventral urethral wall. It will test the null hypothesis that there is no change in striated muscle fiber number, fiber size, or fiber density with age. Material and methods The urethra and its surrounding tissues were removed en bloc at postmortem examination from 34 female cadavers. Twenty-five samples from women with mean age of 52 years (± SD 18; range 15-80 years) were suitable for histologic measurements. Three specimens were from African American women, and the remaining 22 were from white women. Specimens were harvested an average of 15.6 hours (± SD 6.1, range 9-27 hours) after death. Specimens were from consecutive autopsies for which consent for tissue removal was given during days when a member of the investigative team was available. Cadavers with evidence of prior vaginal or incontinence surgeries were excluded. Parity was assessed by review of the patient chart and careful evaluation of the perineum and cervix. Of the 25 women, 14 were found to be parous, 7 were found to be nulliparous, and in 4 individuals parity could not be reliably established. None of the women had a specific neurologic or muscular disease that might have affected the urethral musculature. The specimens were fixed by suspension in 10% buffered formalin and divided in half along the midsagit351
352 Perucchini et al
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Fig 2. Sagittal section of the female urethra with illustration of the measurement strategy. BN, Bladder neck; BW, bladder wall; EM, external meatus; V, vagina.
Fig 1. Components of the striated urogenital sphincter muscle seen after removal of the perineal membrane. CU, Compressor urethrae; US, urethral sphincter; UVS, urethrovaginal sphincter; B, bladder; IR, ischiopubic ramus; SM, smooth muscle; TV, transverse vaginae; U, urethral external meatus; V, vaginal lumen; VW, vaginal wall. (Redrawn from Oelrich.10)
tal plane. One half of the specimen was embedded in paraffin and used to produce a midline sagittal section, 8 µm thick, that included the entire urethra and the bladder base (Fig 2). All sections were stained with use of the Masson trichrome technique on glass slides. A systematic fiber count was obtained at sampling locations corresponding to each decile of the ventral urethra. For the purpose of this study, 0% of urethral length was defined at the external surface of the bladder where the urethra emerged from the bladder and 100% of urethral length was defined at the distal end of the urethral musculature (Fig 2). A line was then drawn perpendicular to the urethral lumen at each decile of urethral length. This line was followed from the ventral surface of the urethra to the dorsal surface, and all striated muscle fibers within consecutive rectangular high-power fields (170 µm 250 µm = 0.0425 mm2, original magnification 40) along this line were counted blinded to the subject’s age. The fiber count was performed without bias induced by the edge effect.11 To compare samples from the same proportion of each urethral length in urethrae of different absolute lengths, the fiber numbers were multiplied by a conversion factor that resulted in exactly 1.25% of urethral length being sampled at each decile (Fig 2). The fibers counted at each decile were then summed to obtain the total num-
ber of sampled fibers. Because, by definition, fibers could be present in nine of the deciles (10%-90%), 11.25% of the total striated sphincter area was actually sampled. The total number of fibers in the entire ventral urethral wall was estimated by multiplying the total number of sampled fibers by 8.89 (100/11.25). Fiber density was then calculated by dividing the total number of sampled fibers by the sampled area as expressed by the sum of the highpower field areas sampled and is expressed as number of fibers per millimeter squared. Fiber diameter was measured from a photomicrograph taken at 25 magnification at each decile. To locate the position along the decile line that would be sampled, a random number table was used. The lesser fiber diameter, defined as the maximum diameter across the lesser aspect of the muscle fiber,12 was then measured for each fiber within the field by using the computer program “NIH Image” (version 1.60). The fiber diameter of at least 200 fibers was measured for every urethra resulting in a total of >10,000 sampled fibers. The total cross-sectional area of the muscle (MAt) was estimated from the total fiber number and the mean muscle fiber area using the following formula: MAt = πr2 Total number of fibers (where r = mean fiber diameter divided by 2). To study the relationship between age and fiber number, a linear regression was used with the model assumptions being checked by using residual inspection. In particular, residuals plotted versus age did not reveal a systematic pattern. The same plot did not indicate heterogeneity of variance of residuals. P < .05 was considered statistically significant. Results The mean total number of fibers within the ventral urethral wall across all 25 urethrae was 17,423 (±SD 9624, range 4788-35,867). For the three young adult nulliparous women (age 15, 21, and 23 years) where least damage
Perucchini et al 353
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Fig 3. Graph showing the decrease of the total fiber number in the ventral urethral wall with age. The squares highlight the fiber number of nulliparous individuals.
Fig 4. Graph illustrating the influence of age on density of fibers in the ventral urethral wall.
Fig 5. Graph showing the correlation between the mean lesser diameter in the striated urogenital sphincter muscle in the ventral urethra and age.
Fig 6. Graph showing correlation between total cross-sectional muscle area in the ventral urethra and age.
would be expected, the mean number of fibers was 33,707 (±SD 3385, range 29,806-35,867). The total number of fibers decreased significantly with age. The regression (Fig 3) shows that on average, over the adult life span, 364 fibers (1%) were lost per year (95% CI 197-531; P < .001, r2 = 0.5). In our sample, the total number of fibers for the specimen with the most fibers (35,867) was >7 times larger than the specimen with the fewest fibers (4788). The mean fiber density was 671 (± SD 296, range 2281374) fibers/mm2 and decreased by 13 fibers/mm2 (2%) per year with age (95% CI 8-17; P < .001), r2 = 0.6) (Fig 4). There was a slightly stronger correlation between fiber density and age (r2 = 0.6) than between total number of fibers and age (r2 = 0.5). Because of weak staining, accurate determination of fiber diameter was not possible for 2 of the 25 samples. Fig 5 shows the distribution of the measured fiber diameters in the urogenital sphincter for 23 samples. The mean fiber diameter was 24 µm (±SD 5, range 4-98 µm) and did not change significantly with age (P = .3). However, when the mean diameters in the sphincter and
compressor muscle were analyzed separately, the mean sphincter muscle fiber diameter was slightly smaller (21.5, ±SD 5.2 µm) than in the compressor muscle (25.1, ±SD 7.2 µm) (P < .001). In the sphincter muscle, the fiber diameter measurements tended to increase with age at a rate of 0.1 µm per year (P < .05), but for the compressor muscle it remained the same. Because fiber number decreased without a corresponding change in fiber diameter, the total crosssectional muscle area in the ventral urethral wall showed a significant decrease with age as shown in Fig 6 (P < .05, r2 = 0.23). Comment This study estimates the total number of muscle fibers in the ventral wall of the striated urogenital sphincter and shows that a loss of urethral striated muscle fibers occurs with aging without a corresponding decrease in fiber size. The resulting loss of contractile tissue is primarily the result of loss of muscle fibers and is manifested by de-
354 Perucchini et al
creased fiber density. The observation that there was no decrease in the mean muscle fiber diameter in the striated urogenital sphincter contrasts with the changes that occur with aging in limb skeletal muscle, where both loss of fibers and atrophy of individual fibers occurs.13 In an earlier study looking at the nerves within a subset of these urethras we found a correlation between the number of nerve fascicles and the number of nerve fibers.14 Whether nerve loss leads to muscle loss or vice versa cannot be determined from these types of evaluations. The ~65% loss in total number of fibers found in this study between the third and the eighth decade of life is consistent with the ~54% loss in urethral closure pressure reported by Rud,9 indicating that these two biologic phenomena are of the same magnitude. Careful direct anatomic-functional correlation will be needed to study this relationship. We believe loss of urethral contractile tissue is likely a contributor to the development of stress incontinence. Recent research has supported a role of the neuromuscular system in stress continence. Blockade of striated muscle activity decreases both resting urethral pressure15,16 and pressure transmission.17 In addition, abnormal nerve function has been demonstrated in stress incontinent women.18 What remains unclear is the way in which this nerve injury would affect urethral function. The findings of the present study, demonstrating significant loss of muscle fibers with aging, is consistent with the hypothesis that muscle loss is a result of nerve injury,19,20 but targeted research will be needed to test this hypothesis. The changes of the striated muscle with age in other areas of the body have been studied extensively. By age 60 to 70 years, overall striated muscle mass decreases by 25% to 30% in humans,21 with increasing muscle fiber loss beginning after age 50 to 60 years. With a decrease in physical activity, as occurs with immobilization, muscle atrophy results primarily from a decrease in the cross-sectional area of individual fibers. The muscle atrophy associated with decreased activity is reversible, and muscle mass and cross-sectional area are restored when normal activity levels are restored, even in individuals in their 90s.22 In contrast, loss of muscle with aging results from both the loss of muscle fibers and a decrease in individual fiber diameter. The relative importance of these effects is probably highly dependent on the muscle and fiber types that are studied. Atrophy of individual muscle fibers appears to be a more significant factor for type II than for type I fibers. There are some data to suggest that more severe fiber loss in skeletal muscle may result in slight hypertrophy of type I fibers.13 The striated sphincter has been shown to be composed largely of slow twitch (type I) fibers,23 but there is some evidence that the rhabdosphincter is composed of both fast- and slow-twitch fibers in women.24,25 In addition to intrinsic age-associated loss of muscle
March 2002 Am J Obstet Gynecol
fibers in the urethral striated sphincter, birth trauma may also contribute to muscle loss. The latter could be responsible for the large variation in the magnitude of the fiber loss seen in the present study. It is noteworthy that the data on fiber number from five of the six urethrae from women known to be nulliparous lay above the regression line. The cause of muscle loss is an important topic for future study. Birth trauma could be responsible for the muscle fiber loss observed in some of the younger individuals. That fiber loss may be caused by irreparable damage to the fibers, a permanent loss of contact between the nerves and the muscle fibers, or axonal loss. The degree of variation between the specimens is striking and must be considered when analyzing summary statistics. For example, despite the systematic decrease in average fiber number observed with age, there was a 78-year-old woman with a total of 24,288 fibers, a value similar to the 29,806 fibers found in the 23-year-old woman and very different from others of similar age. It is interesting to note that this older woman was nulliparous, suggesting that fiber loss may indeed be associated with parity. Despite the attractiveness of this hypothesis, our sample did not include a sufficient number of older nulliparous individuals to evaluate parity as a determinant of muscle loss. Comparison of fiber numbers in age-matched nulliparous and parous women will be needed to establish the relative importance of age and parity. The differences in total muscle cross-sectional area resulting from the several-fold range of fiber numbers are consistent with the real variation in urethral closure pressure found clinically.2 Anatomic research has its limitations. The lack of reliable continence information for these cadavers precludes our ascertaining the relationship between histologic features of the muscle and continence. In addition, artifacts may exist because of postmortem changes. There may be some shrinkage artifact caused by fixation and loss of vascular tone, but the number of muscle fibers should not change. When viewed with these limitations, we believe that the insights gained from these anatomic investigations provide useful information that cannot be gained in other ways. We have chosen to examine the striated urogenital sphincter in the ventral wall because all elements of this muscle are represented in this region. The compressor urethrae and urethrovaginal sphincter diverge from the wall of the urethra along its lateral margin and are, therefore, not fully represented in the lateral wall and are often missing in the dorsal wall. The loss of urethral striated muscle fibers provides insight into the biologic process of urethral deterioration and should help guide further basic and clinical studies to more clearly elucidate the alterations found in stress urinary incontinence. The relative importance of urethral damage and loss of urethral support remains to be clarified.
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REFERENCES
1. Ala-Ketola L. Roentgen diagnosis of female stress urinary incontinence. Acta Obstet Gynecol Scand 1973;23(Suppl):259. 2. Hilton P, Stanton SL. Urethral pressure measurement by microtransducer: the results in symptom-free women and in those with genuine stress incontinence. Br J Obstet Gynaecol 1983;90:919-33. 3. Smith ARB, Hosker GL, Warrell DW. The role of partial denervation of the pelvic floor in the aetiology of genitourinary prolapse and stress incontinence of urine: a neurophysiologic study. Br J Obstet Gynaecol 1989;96:24-8. 4. Richardson AC, Edmonds PB, Williams NL. Treatment of stress urinary incontinence due to paravaginal defect. Obstet Gynecol 1981;57:357-62. 5. Klutke GC, Golomb J, Barbaric Z, Raz S. The anatomy of stress incontinence: magnetic resonance imaging of the female bladder neck. J Urol 1990;143:463-6. 6. Huddleston HT, Dunnihoo DR, Huddleston PM. Magnetic resonance imaging of defects in DeLancey’s vaginal support levels I, II, and III. Am J Obstet Gynecol 1995;172:1778-82. 7. Carlile A, Davies I, Rigby A, Brocklehurst JC. Age changes in the human female urethra: a morphometric study. J Urol 1988; 139:532-5. 8. Huisman AB. Aspects on the anatomy of the female urethra with special relation to urinary continence. Contrib Gynecol Obstet 1983;10:1-31. 9. Rud T. Urethral pressure profile in continent women from childhood to old age. Acta Obstet Gynecol Scand 1980;59:331-5. 10. Oelrich TM. The striated urogenital sphincter muscle in the female. Anat Rec 1983;205:223-32. 11. Gundersen HJG. Estimators of the number of objects per area unbiased by edge effects. Microsc Acta 1978;81:107-17. 12. Bennington JL, Krupp M. Morphometric analysis of muscle. In: Heffner RR Jr, editor. Muscle pathology. New York: Churchill Livingstone; 1984. p. 43-71. 13. Lexell J, Taylor C, Sjostrom M. What is the cause of the ageing atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15-to 83-year-old men. J Neurol Sci 1988;84:275-94.
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14. Pandit M, DeLancey JO, Ashton-Miller JA, Iyengar J, Blaivas M, Perucchini D. Quantification of intramuscular nerves within the female striated urogenital sphincter muscle. Obstet Gynecol 2000;95:797-800. 15. Thind P, Bagi P, Mieszczak C, Lose G. Influence of pudendal nerve blockade on stress relaxation in the female urethra. Neurourol Urodynam 1996;15:31-6. 16. Rud T, Anderson KE, Asmussen M, Hunting A, Ulmsten U. Factors maintaining the intraurethral pressure in women. Invest Urol 1980;17:343-7. 17. Bump RC, Huang KC, McClish DK, Fantl JA. Effect of narcotic anesthesia and skeletal muscle paralysis on passive and dynamic urethral function of stress continent and incontinent women. Neurourol Urodynam 1991;10:523-32. 18. Snooks SJ, Badenoch DF, Tiptaft RC, Swash M. Perineal nerve damage in genuine stress urinary incontinence. Br J Urol 1985;57:422-6. 19. Lewis DM. The effect of denervation on the mechanical and electrical responses of fast and slow mammalian twitch muscle. J Physiol 1972;222:51-75. 20. Al-Amood SW, Lewis DM, Schmalbruch H. Effects of chronic electrical stimulation on contractile properties of long-term denervated rat skeletal muscle. J Physiol 1991;441:243-56. 21. Grimby G, Saltin B. The ageing muscle. Clin Phys 1983; 3:209-18. 22. Fiatarone MA, Marks EC, Ryan ND, Meredith CN, Lipsitz LA, Evans WJ. High-intensity strength training in nonagenarians. JAMA 1990;263:3029-34. 23. Gosling JA, Dixon JS, Critchley HOD, Thompson S-A. A comparative study of the human external sphincter and periurethral levator ani muscles. Br J Urol 1981;53:35-41. 24. Schroder HD, Reske-Nielsen E. Fiber types in the striated urethral and anal sphincters. Acta Neuropathol (Berl) 1983; 60:278-82. 25. Tokunaka S, Murakami U, Fujii H, Okamura K, Miyata M, Hashimoto H, et al. Coexistence of fast and slow myosin isozymes in human external urethral sphincter: a preliminary report. J Urol 1987;138:659-62.
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Loss of urethral support and decreased urethral function can result in stress urinary incontinence in women.1-3 Although several authors have described the anatomic defects responsible for urethral hypermobility,4-6 the anatomic changes responsible for decreased urethral closure pressure have received less attention.7,8 In the female urethra, closure pressure is known to be developed principally by contraction of the smooth and striated muscle. Because a decrease in muscle cells might explain the observed closure pressure decrease with age,2,9 we hypothesized that a study of striated muscle histomorphometry might give useful insights into the underlying mechanisms of urethral dysfunction. All elements of the striated urogenital sphincter muscle are found in the ventral urethral wall. The sphincter urethrae, the urethrovaginal sphincter, and the compressor urethrae are all present in the ventral wall, whereas the dorsal wall contains only the sphincter urethrae because the urethrovaginal sphincter and the compressor urethrae diverge from the urethral wall to From the Department of Obstetrics and Gynecology, University of Michigan, and the Department Frauenheilkunde, Universitätspital Zurich. Supported by a grant from the Swiss National Science Foundation and a National Institutes of Health Grant R01 DK 47516-03. Received for publication February 15, 2001; revised June 6, 2001; accepted October 2, 2001. Reprint requests: Daniele Perucchini, MD, Departement Frauenheilkunde, Universitatsspital Zurich, Frauenklinikstrasse 10, CH-8091 Zurich, Switzerland. E-mail: [email protected] Copyright 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/121089 doi:10.1067/mob.2002.121089
pass laterally toward the ischiopubic ramus and the vaginal wall (Fig 1).10 This study focuses on the striated urogenital muscle layer in the ventral urethral wall. It will test the null hypothesis that there is no change in striated muscle fiber number, fiber size, or fiber density with age. Material and methods The urethra and its surrounding tissues were removed en bloc at postmortem examination from 34 female cadavers. Twenty-five samples from women with mean age of 52 years (± SD 18; range 15-80 years) were suitable for histologic measurements. Three specimens were from African American women, and the remaining 22 were from white women. Specimens were harvested an average of 15.6 hours (± SD 6.1, range 9-27 hours) after death. Specimens were from consecutive autopsies for which consent for tissue removal was given during days when a member of the investigative team was available. Cadavers with evidence of prior vaginal or incontinence surgeries were excluded. Parity was assessed by review of the patient chart and careful evaluation of the perineum and cervix. Of the 25 women, 14 were found to be parous, 7 were found to be nulliparous, and in 4 individuals parity could not be reliably established. None of the women had a specific neurologic or muscular disease that might have affected the urethral musculature. The specimens were fixed by suspension in 10% buffered formalin and divided in half along the midsagit351
352 Perucchini et al
March 2002 Am J Obstet Gynecol
Fig 2. Sagittal section of the female urethra with illustration of the measurement strategy. BN, Bladder neck; BW, bladder wall; EM, external meatus; V, vagina.
Fig 1. Components of the striated urogenital sphincter muscle seen after removal of the perineal membrane. CU, Compressor urethrae; US, urethral sphincter; UVS, urethrovaginal sphincter; B, bladder; IR, ischiopubic ramus; SM, smooth muscle; TV, transverse vaginae; U, urethral external meatus; V, vaginal lumen; VW, vaginal wall. (Redrawn from Oelrich.10)
tal plane. One half of the specimen was embedded in paraffin and used to produce a midline sagittal section, 8 µm thick, that included the entire urethra and the bladder base (Fig 2). All sections were stained with use of the Masson trichrome technique on glass slides. A systematic fiber count was obtained at sampling locations corresponding to each decile of the ventral urethra. For the purpose of this study, 0% of urethral length was defined at the external surface of the bladder where the urethra emerged from the bladder and 100% of urethral length was defined at the distal end of the urethral musculature (Fig 2). A line was then drawn perpendicular to the urethral lumen at each decile of urethral length. This line was followed from the ventral surface of the urethra to the dorsal surface, and all striated muscle fibers within consecutive rectangular high-power fields (170 µm 250 µm = 0.0425 mm2, original magnification 40) along this line were counted blinded to the subject’s age. The fiber count was performed without bias induced by the edge effect.11 To compare samples from the same proportion of each urethral length in urethrae of different absolute lengths, the fiber numbers were multiplied by a conversion factor that resulted in exactly 1.25% of urethral length being sampled at each decile (Fig 2). The fibers counted at each decile were then summed to obtain the total num-
ber of sampled fibers. Because, by definition, fibers could be present in nine of the deciles (10%-90%), 11.25% of the total striated sphincter area was actually sampled. The total number of fibers in the entire ventral urethral wall was estimated by multiplying the total number of sampled fibers by 8.89 (100/11.25). Fiber density was then calculated by dividing the total number of sampled fibers by the sampled area as expressed by the sum of the highpower field areas sampled and is expressed as number of fibers per millimeter squared. Fiber diameter was measured from a photomicrograph taken at 25 magnification at each decile. To locate the position along the decile line that would be sampled, a random number table was used. The lesser fiber diameter, defined as the maximum diameter across the lesser aspect of the muscle fiber,12 was then measured for each fiber within the field by using the computer program “NIH Image” (version 1.60). The fiber diameter of at least 200 fibers was measured for every urethra resulting in a total of >10,000 sampled fibers. The total cross-sectional area of the muscle (MAt) was estimated from the total fiber number and the mean muscle fiber area using the following formula: MAt = πr2 Total number of fibers (where r = mean fiber diameter divided by 2). To study the relationship between age and fiber number, a linear regression was used with the model assumptions being checked by using residual inspection. In particular, residuals plotted versus age did not reveal a systematic pattern. The same plot did not indicate heterogeneity of variance of residuals. P < .05 was considered statistically significant. Results The mean total number of fibers within the ventral urethral wall across all 25 urethrae was 17,423 (±SD 9624, range 4788-35,867). For the three young adult nulliparous women (age 15, 21, and 23 years) where least damage
Perucchini et al 353
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Fig 3. Graph showing the decrease of the total fiber number in the ventral urethral wall with age. The squares highlight the fiber number of nulliparous individuals.
Fig 4. Graph illustrating the influence of age on density of fibers in the ventral urethral wall.
Fig 5. Graph showing the correlation between the mean lesser diameter in the striated urogenital sphincter muscle in the ventral urethra and age.
Fig 6. Graph showing correlation between total cross-sectional muscle area in the ventral urethra and age.
would be expected, the mean number of fibers was 33,707 (±SD 3385, range 29,806-35,867). The total number of fibers decreased significantly with age. The regression (Fig 3) shows that on average, over the adult life span, 364 fibers (1%) were lost per year (95% CI 197-531; P < .001, r2 = 0.5). In our sample, the total number of fibers for the specimen with the most fibers (35,867) was >7 times larger than the specimen with the fewest fibers (4788). The mean fiber density was 671 (± SD 296, range 2281374) fibers/mm2 and decreased by 13 fibers/mm2 (2%) per year with age (95% CI 8-17; P < .001), r2 = 0.6) (Fig 4). There was a slightly stronger correlation between fiber density and age (r2 = 0.6) than between total number of fibers and age (r2 = 0.5). Because of weak staining, accurate determination of fiber diameter was not possible for 2 of the 25 samples. Fig 5 shows the distribution of the measured fiber diameters in the urogenital sphincter for 23 samples. The mean fiber diameter was 24 µm (±SD 5, range 4-98 µm) and did not change significantly with age (P = .3). However, when the mean diameters in the sphincter and
compressor muscle were analyzed separately, the mean sphincter muscle fiber diameter was slightly smaller (21.5, ±SD 5.2 µm) than in the compressor muscle (25.1, ±SD 7.2 µm) (P < .001). In the sphincter muscle, the fiber diameter measurements tended to increase with age at a rate of 0.1 µm per year (P < .05), but for the compressor muscle it remained the same. Because fiber number decreased without a corresponding change in fiber diameter, the total crosssectional muscle area in the ventral urethral wall showed a significant decrease with age as shown in Fig 6 (P < .05, r2 = 0.23). Comment This study estimates the total number of muscle fibers in the ventral wall of the striated urogenital sphincter and shows that a loss of urethral striated muscle fibers occurs with aging without a corresponding decrease in fiber size. The resulting loss of contractile tissue is primarily the result of loss of muscle fibers and is manifested by de-
354 Perucchini et al
creased fiber density. The observation that there was no decrease in the mean muscle fiber diameter in the striated urogenital sphincter contrasts with the changes that occur with aging in limb skeletal muscle, where both loss of fibers and atrophy of individual fibers occurs.13 In an earlier study looking at the nerves within a subset of these urethras we found a correlation between the number of nerve fascicles and the number of nerve fibers.14 Whether nerve loss leads to muscle loss or vice versa cannot be determined from these types of evaluations. The ~65% loss in total number of fibers found in this study between the third and the eighth decade of life is consistent with the ~54% loss in urethral closure pressure reported by Rud,9 indicating that these two biologic phenomena are of the same magnitude. Careful direct anatomic-functional correlation will be needed to study this relationship. We believe loss of urethral contractile tissue is likely a contributor to the development of stress incontinence. Recent research has supported a role of the neuromuscular system in stress continence. Blockade of striated muscle activity decreases both resting urethral pressure15,16 and pressure transmission.17 In addition, abnormal nerve function has been demonstrated in stress incontinent women.18 What remains unclear is the way in which this nerve injury would affect urethral function. The findings of the present study, demonstrating significant loss of muscle fibers with aging, is consistent with the hypothesis that muscle loss is a result of nerve injury,19,20 but targeted research will be needed to test this hypothesis. The changes of the striated muscle with age in other areas of the body have been studied extensively. By age 60 to 70 years, overall striated muscle mass decreases by 25% to 30% in humans,21 with increasing muscle fiber loss beginning after age 50 to 60 years. With a decrease in physical activity, as occurs with immobilization, muscle atrophy results primarily from a decrease in the cross-sectional area of individual fibers. The muscle atrophy associated with decreased activity is reversible, and muscle mass and cross-sectional area are restored when normal activity levels are restored, even in individuals in their 90s.22 In contrast, loss of muscle with aging results from both the loss of muscle fibers and a decrease in individual fiber diameter. The relative importance of these effects is probably highly dependent on the muscle and fiber types that are studied. Atrophy of individual muscle fibers appears to be a more significant factor for type II than for type I fibers. There are some data to suggest that more severe fiber loss in skeletal muscle may result in slight hypertrophy of type I fibers.13 The striated sphincter has been shown to be composed largely of slow twitch (type I) fibers,23 but there is some evidence that the rhabdosphincter is composed of both fast- and slow-twitch fibers in women.24,25 In addition to intrinsic age-associated loss of muscle
March 2002 Am J Obstet Gynecol
fibers in the urethral striated sphincter, birth trauma may also contribute to muscle loss. The latter could be responsible for the large variation in the magnitude of the fiber loss seen in the present study. It is noteworthy that the data on fiber number from five of the six urethrae from women known to be nulliparous lay above the regression line. The cause of muscle loss is an important topic for future study. Birth trauma could be responsible for the muscle fiber loss observed in some of the younger individuals. That fiber loss may be caused by irreparable damage to the fibers, a permanent loss of contact between the nerves and the muscle fibers, or axonal loss. The degree of variation between the specimens is striking and must be considered when analyzing summary statistics. For example, despite the systematic decrease in average fiber number observed with age, there was a 78-year-old woman with a total of 24,288 fibers, a value similar to the 29,806 fibers found in the 23-year-old woman and very different from others of similar age. It is interesting to note that this older woman was nulliparous, suggesting that fiber loss may indeed be associated with parity. Despite the attractiveness of this hypothesis, our sample did not include a sufficient number of older nulliparous individuals to evaluate parity as a determinant of muscle loss. Comparison of fiber numbers in age-matched nulliparous and parous women will be needed to establish the relative importance of age and parity. The differences in total muscle cross-sectional area resulting from the several-fold range of fiber numbers are consistent with the real variation in urethral closure pressure found clinically.2 Anatomic research has its limitations. The lack of reliable continence information for these cadavers precludes our ascertaining the relationship between histologic features of the muscle and continence. In addition, artifacts may exist because of postmortem changes. There may be some shrinkage artifact caused by fixation and loss of vascular tone, but the number of muscle fibers should not change. When viewed with these limitations, we believe that the insights gained from these anatomic investigations provide useful information that cannot be gained in other ways. We have chosen to examine the striated urogenital sphincter in the ventral wall because all elements of this muscle are represented in this region. The compressor urethrae and urethrovaginal sphincter diverge from the wall of the urethra along its lateral margin and are, therefore, not fully represented in the lateral wall and are often missing in the dorsal wall. The loss of urethral striated muscle fibers provides insight into the biologic process of urethral deterioration and should help guide further basic and clinical studies to more clearly elucidate the alterations found in stress urinary incontinence. The relative importance of urethral damage and loss of urethral support remains to be clarified.
Volume 186, Number 3 Am J Obstet Gynecol
REFERENCES
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