Histomorphometry of penile smooth muscle fiber in severe erectile dysfunction

ABSTRACT CONTEXT AND OBJECTIVE: Smooth muscle fiber has fundamental importance in erection. Alterations in its function or quantity may be associated with erectile dysfunction. The study objective was to assess the proportion of penile smooth muscle fiber in patients with severe erectile dysfunction. DESIGN AND SETTING: Clinical study, in the Sexual Dysfunction Group, Universidade Federal de São Paulo (Unifesp), and in the Anatomy Laboratory, Universidade Estadual do Rio de Janeiro (UERJ). METHODS: Twenty patients with severe erectile dysfunction were selected to form two groups of ten patients: one with normal arterial flow (age range: 44 to 78 years) and the other with altered arterial flow (age range: 38 to 67 years). These groups were compared with a group formed by ten cadavers aged 18 to 25 years that were presumed to have been potent. Quantification of the smooth muscle fibers was done by means of an immunohistochemical study. RESULTS: The proportion of smooth muscle fiber found was 41.15% for the control group. The patients with erectile dysfunction and normal arterial flow presented 27.24% and those with altered arterial flow presented 25.74%; 19 patients presented at least one chronic disease or risk factor for erectile dysfunction, with prominence for diabetes mellitus, systemic arterial hypertension and smoking. CONCLUSION: Among patients with severe erectile dysfunction, the arterial flow on its own does not present interference in the proportion of smooth muscle fiber. The diminution of the proportion of smooth muscle fiber may result from chronic diseases and vascular risk factors.


METHODS
The sample for the study consisted of 10 cadavers of young men and 20 patients with erectile dysfunction, distributed into the following three groups: • Group I (control): ten cadavers of young men; • Group II: ten patients with erectile dysfunction and normal arterial flow as seen on duplex ultrasound; • Group III: ten patients with erectile dysfunction and arterial insufficiency as seen on duplex ultrasound.
All patients signed an informed consent statement, and the study received the approval of the research ethics committee of Universidade Federal de São Paulo.
The material for group I was formed by specimens of the corpus cavernosum of the penis shaft from cadavers aged between 18 and 25 years.These individuals had suffered violent death caused by car accidents, murder by stabbing or shooting, or falling from a height.The specimens were obtained within 12 hours of death.Cadavers were excluded if these individuals had died of chronic diseases, poisoning or drowning, or if they presented injuries due to physical agents, heat or chemical substances, or if they presented genital lesions.The genitalia were examined to search for the excluding criteria of fibrotic plaque and other alterations, abdominal scars suggesting previous surgery, absence of testicles or testicular volume not in keeping with the individual's age.Cadavers were also excluded if they did not present secondary sexual characteristics compatible with male sex and the individual's age.Therefore, despite the impossibility of being sure that the cadavers were of potent men, the above criteria enable the assumption that they actually were potent.The necropsies were performed at the Instituto Médico Legal (Medical Examiners' Institute) of the State of São Paulo during November and December 1997.METHODS: Twenty patients with severe erectile dysfunction were selected to form two groups of ten patients: one with normal arterial flow (age range: 44 to 78 years) and the other with altered arterial flow (age range: 38 to 67 years).These groups were compared with a group formed by ten cadavers aged 18 to 25 years that were presumed to have been potent.Quantification of the smooth muscle fibers was done by means of an immunohistochemical study.

RESULTS:
The proportion of smooth muscle fiber found was 41.15% for the control group.The patients with erectile dysfunction and normal arterial flow presented 27.24% and those with altered arterial flow presented 25.74%; 19 patients presented at least one chronic disease or risk factor for erectile dysfunction, with prominence for diabetes mellitus, systemic arterial hypertension and smoking.
CONCLUSION: Among patients with severe erectile dysfunction, the arterial flow on its own does not present interference in the proportion of smooth muscle fiber.The diminution of the proportion of smooth muscle fiber may result from chronic diseases and vascular risk factors.Groups II and III were formed by selecting patients who were attending the outpatient clinic of the erectile dysfunction sector of Hospital São Paulo of the Universidade Federal de São Paulo -Escola Paulista de Medicina (Unifesp-EPM), between February and September 1998.A detailed clinical examination was also performed on each patient, with special attention to underlying diseases and risk factors.The systemic arterial pressure levels were evaluated, and the genitalia were examined in a search for alterations that could exclude them from the study, such as fibrotic plaque or deformities.Blood was collected for performing the following laboratory tests: fasting glycemia, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, total testosterone and prolactin (as seen in Table 4).
The International Index of Erectile Function (IIEF) was applied. 7Patients were selected if they had had a complaint of erectile dysfunction for at least one year and if their total score in the erectile dysfunction domain of the IIEF (questions 1, 2, 3, 4, 5 and 15) was a maximum of 6, representing a severe degree of erectile dysfunction. 8ext, the patients were submitted to the drug-induced erection test, 9 using 10 µg of PGE1 injected into the corpus cavernosum.The quality of the erection was graded according to the following criteria: 0. no tumescence I. penile tumescence insufficient for penetration II.penile tumescence sufficient for penetration III.full erection Patients were included in the study if the drug-induced erection test showed they would be unable to achieve penetration, i.e. they were graded 0 or I.
Finally, the patients were submitted to duplex ultrasound for assessing the systolic peak velocity.From this, two study groups were formed: group II (patients with normal systolic peak velocity) and group III (patients with arterial insufficiency).][12][13][14] Patients with systolic peak between 25 and 30 cm/s were excluded.In the end, groups II and III were each composed of 10 patients.
Among these patients, specimens from the corpus cavernosum of the penis shaft were obtained under local anesthetic or during surgery for the implantation of a penile prosthesis.
Three sections were made per specimen.The first was stained using the hematoxylineosin method for a general analysis of the tissue (Figure 1-A).The second was stained using the Masson trichrome method for identifying the muscle and conjunctive tissue (Figure 1-B).
The third section was studied by means of the immunohistochemical reaction, using the avidin-biotin complex method.From the avidin-biotin method, sections were considered to be positive when they presented smooth muscle fiber in tones of chestnut brown, contrasting with a blue background.Differences between the structures within the corpus cavernosum (smooth muscle and collagen tissue) could thus be accentuated (Figure 2).
To quantify the smooth muscle fiber, each specimen was evaluated in 15 distinct fields.The laminae were analyzed under an optical microscope at a magnification of 400 x.This was done according to the principles of image segmentation and subtraction. 15,16The original colors of the images were maintained and transformed into graphs with the colors represented by the hue, saturation and luminosity (HSL).The average value from the 15 measurements was considered to be the proportion of smooth muscle fiber for each penis (Figure 3).

RESULTS
Out of a total of 146 patients with complaints of erectile dysfunction who were at-Figure 2. Photomicrograph of the corpus cavernosum with detection of smooth muscle fiber using immunohistochemical analysis (anti-smooth muscle antibodies), 400 x magnification.group I was 21.6 years, and thus significantly younger than in the two groups of patients with erectile dysfunction: 63 years in group II and 56.2 years in group III, which were comparable with each other.Despite the significant age differences between the groups, we chose this protocol in a attempt to ensure that the cadavers were of potent men.
The parameters of hue and saturation were considered in order to distinguish the smooth muscle tissue from the other structures.Thus, it was ensured that the evaluation would be restricted just to the smooth muscle fibers that had previously been immunologically marked (tones of chestnutbrown color), thereby eliminating the other structures (tones of blue).The proportion was calculated from the area occupied by the smooth muscle fiber in relation to the total area of the field under analysis. 15,16gure 3. (A) Photomicrograph of the corpus cavernosum subjected to immunohistochemical analysis using anti-smooth muscle antibodies, 400 x magnification.The same histological section during the quantification of smooth muscle fiber: photograph of the image on the computer monitor screen.tended, 28 patients (21.91%) met the inclusion criteria.However, eight patients with systolic peak velocity of more than 30 cm/s were lost from the follow-up.Thus, the final composition of groups II and III was 10 patients each.
Table 1 presents the ages and causes of death in group I (the cadavers of young men).In this group the ages ranged from 18 to 25 years (average of 21.6 years).Among the patients (Table 2), the ages ranged from 44 to 78 years (average of 63 years) in group II (normal arterial flow), and from 38 to 67 years (average of 56.2 years) in group III (arterial insufficiency).
Table 2 also shows the length of time for which the patients had had their complaint, which ranged from 1 to 20 years in group II (average of 5.1 years) and from 1 to 10 years in group III (average of 5.2 years), and the grade of erectile dysfunction according to the International Index of Erectile Function (IIEF), which ranged from 2 to 6 points in group II (average of 4.9 points) and from 1 to 6 points in group III (average of 4.7 points).
The frequencies of associated diseases and risk factors and the laboratory test results for groups II and III are presented in Tables 3 and  4, respectively.
Table 5 presents the proportion of smooth muscle fiber in the three groups.The range was from 28.92% to 47.63% in group I (average of 41.15%), from 22.16% to 35.51% in group II (average of 27.24%) and from 22.03% to 29.13% in group III (average of 25.74%).

DISCUSSION
Muscle tonus regulates the erection, detumescence and flaccidity of the penis.Consequently, the functional and quantitative integrity of the smooth muscle fiber is fundamental for the mechanism of erection. 17There may be a decrease in the quantity of smooth muscle fiber with advancing age, with a proportion of as little as 25 to 30%. 18of this structure remaining at the age of 75 years.Thus, a study of cadavers without evidence of penile injury found that the quantity of smooth muscle fiber varied as follows: 57% in individuals aged 20 to 40 years, 54% at ages between 40 and 50 years, 51% between 50 and 60 years, 48% between 60 and 70 years and, finally, 44% at ages of between 70 and 80 years. 18On the other hand, there are reports giving contrary information, showing that there was no significant difference in the proportion of smooth muscle fiber between individuals without erectile dysfunction and without chronic diseases, comparing two distinct age groups. 19n the present study, the average age in     Biopsies performed on the corpus cavernosum also allow the smooth muscle fiber to be quantified.One study found a proportion of smooth muscle fiber of 34% in normal individuals and 30.4% in patients with erectile dysfunction, a difference that was not significant. 20n the other hand, the proportion of smooth muscle fiber may reach 40 to 52% in normal men, while being 10 to 36% in individuals with venous-occlusive dysfunction and 13 to 25% in patients with arterial disease. 19alues more similar to ours were found in a study that compared the proportion of smooth muscle fiber of the corpus cavernosum in normal individuals and in those with erectile dysfunction. 21In this study, the average values were 50% (range: 41 to 61%) and 31% (range: 22 to 46%), respectively.In another study, 22 the proportions of smooth muscle fiber found in normal men and those with erectile dysfunction were 41.78% for normal individuals, 28.08% for men with erectile dysfunction of venous cause and 23.90% for those with arterial insufficiency.The differences between the three categories presented statistical significance.It was concluded that the ischemic damage would be more severe in patients with arterial dysfunction, thereby causing a more accentuated decrease in smooth muscle tissue.
In the present study, the proportions of smooth muscle fiber found for each group were 41.15% in group I (control), 27.24% in group II (normal arterial flow) and 25.74% for group III (arterial insufficiency).The difference between the group taken to be the control and the patients with erectile dysfunction was statistically significant (p < 0.0001).
The possible mechanisms implicated in the diminution of the quantity of smooth muscle fiber have been studied.In fact, the oxygen pressure within the corpus cavernosum during erection has been found to be lower in patients with severe arteriopathy or diabetic myopathy of the corpus cavernosum who did not respond to prostaglandin injection, despite increased blood inflow. 23Furthermore, in such cases, computerized planimetry showed decreased quantities of smooth muscle tissue in the corpus cavernosum. 24t has recently been observed 24 that the oxygen pressure within the corpus cavernosum increases from 25 to 40 mmHg in the flaccid state to 90 to 100 mmHg during erection.Alterations in oxygen levels are thought to be implicated in imbalance between the quantities of smooth muscle fiber and connective tissue.Such balance is fundamental for enabling erection to take place, since it is the connective tissue (collagen) that causes the erection to be maintained.On the other hand, increased collagen associated with diminished smooth muscle fiber leads to an incapability to achieve venous occlusion. 23,24he decrease in the proportion of smooth muscle fiber may also be caused by the simultaneous presence of chronic diseases that are associated with erectile dysfunction, such as hypertension, diabetes, coronary insufficiency and chronic renal insufficiency, 25,26 or by several risk factors like smoking 27 or hypercholesterolemia. 28 Of the 20 patients that we studied, 19 presented at least one morbid condition, and thus only one patient, who was in group II (normal arterial flow), did not present any chronic disease.We therefore did not find any difference between groups II and III, in relation to chronic diseases.Diabetes mellitus was the most  frequent finding, affecting six patients in group II and seven in group III.This was followed by systemic arterial hypertension, which affected six patients in group II and five in group III.The other diseases had lesser frequency.Three patients in group II and seven in group III were smokers.Smoking may be related to greater incidence of arterial insufficiency, although no relation has been found with the proportion of smooth muscle fiber.
The assays of total cholesterol and lowdensity lipoprotein (LDL) in cholesterol in the present study presented significantly greater values in group III (arterial insufficiency).The total cholesterol levels were 167.8 ± 33.24 mg/dl and 218.8 ± 60.88 mg/dl, respectively, in groups II and III.The LDL cholesterol presented values of 102.5 ± 27.66 mg/dl for group II and 146.4 ± 26.5 mg/dl for group III.In our patients, the increased cholesterol levels were associated with arterial insufficiency.The other laboratory tests performed did not present any significant differences between the groups.

CONCLUSION
We can conclude that the proportion of smooth muscle fiber found in our control group, with values of 40 to 50%, was within the range of normality.On the other hand, there was a marked decrease in the quantity of smooth muscle fiber among the patients with severe erectile dysfunction.
Thus, it would seem to be correct to suggest that greater care taken with patients who are at risk may diminish the possibility of major irreversible injury to erectile tissue, through the prevention and treatment of conditions that are recognized to have an association with erectile dysfunction.

Age:
Variance analysis by Kruskal-Wallis ranking; p < 0.0001; I < II and III.Length of complaint: Mann-Whitney Test; Group II x Group III; Group II = Group III.

Table 3 .
Frequency of associated diseases and risk factors in erectile dysfunction groups, via Fisher's exact test (p) or chi-squared test (χ 2 )

Table 1 .
Characteristics of the cadavers (control group) studied

Table 2 .
Ages (in years), length of time with complaint and grade of erectile dysfunction (ED) among the patients, according to the International Index of Erectile Function (IIEF)

Table 4 .
Results from the laboratory tests for erectile dysfunction groups (via the Mann-Whitney test) SD = standard deviation; HDL = high-density lipoprotein; LDL = low-density lipoprotein; * = significant.

Table 5 .
Proportions of smooth muscle fiber in cadavers and erectile dysfunction patients with normal arterial flow and with arterial insufficiency Variance analysis by Kruskal-Wallis ranking; p < 0.0001 *; I > II and III.