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Association of elevated estradiol with remote testicular trauma in young infertile men
Urology-andrology Vol. 62, No.1, July 1994
FERTILITY AND STERILITY Copyright
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Printed on acid-free paper in U. S. A.
1994 The American Fertility Society
Association of elevated estradiol with remote testicular trauma in young infertile men
Wolfram E. Nolten, M.D.*t Sharon P. Viosca, B.S.:\: Stanley G. Korenman, M.D.:\:
Roya Mardi, B.A.:\: Sander S. Shapiro, M.D.§
University of Wisconsin, Madison, Wisconsin, and University of California Los Angeles (UCLA) Medical Center, Los Angeles, California
Objective: To determine the incidence of remote testicular trauma and of possible related permanent hormonal and seminal changes in infertile men. Design: Retrospective clinical study of hormonal and seminal parameters in a subpopulation of infertile men. Setting: Andrology Clinic in an academic research environment. Participants: Infertile men, with and without history of remote testicular trauma, and fertile volunteers. Main Outcome Measures: Percentage of infertile men with history of blunt testicular trauma, concentrations of reproductive hormones, and semen parameters. Results: Significant remote blunt testicular trauma was reported by 16.8% of infertile men. This had occurred 2 to 17 years (mean, 16.4 years) before evaluation, mostly with contact sports at adolescent age. Estradiol concentrations after testicular trauma were 19% and 25% higher than in infertile men without history of testicular injury and in fertile controls. Elevated E2 levels did not correlate with T. Infertile men with and without history of testicular trauma showed changes in seminal parameters. Conclusion: The incidence of remote blunt testicular trauma in infertile men is unexpectedly high. After injury, FSH-stimulated aromatization ofT may increase testicular E2 production, interfere with spermatogenesis, and cause infertility. Consistent use of protective devices in contact sports is recommended. Fertil Steril 1994;62:143-9 Key Words: Testicular trauma, E 2, male infertility
Blunt testicular trauma is not considered as a common cause of impaired spermatogenesis and altered testicular endocrine function (1, 2). Longterm hormonal consequences of testicular trauma have not been reported so far. Adolescent boys encounter a significant risk of testicular injury when Received August 30, 1993; revised and accepted March 9, 1994. * Reprint requests: Wolfram E. Nolten, M.D., Department of Medicine, University of Wisconsin Hospital and Clinics, H4/ 564 CSC, 600 Highland Avenue, Madison, Wisconsin 537925148 (FAX: 608-262-9289). t Department of Medicine, University of Wisconsin. :j: Department of Medicine, UCLA Medical Center. § Department of Obstetrics and Gynecology, University of Wisconsin. Vol. 62, No.1, July 1994
participating in contact sports. Protective cups enclose the testes but are often not used, and for many athletic activities no protection is required. Demonstration of permanent changes in testicular function caused by testicular trauma would support the need for better and more consistent protection of the young athlete. The evaluation of a large population of young adult men for suspected infertility offered an opportunity to study the prevalence of past testicular trauma in this group and to investigate the possibility that abnormalities in testicular function in adult life could be related to remote genital injury. To assess this possibility, we inquired with all male infertility patients about a possible history oftesticular trauma. Semen parameters and reproductive Nolten et al.
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143
hormone concentrations in infertile males who had experienced testicular trauma in the past were compared with those in infertile men without a history of testicular injury and with those in fertile men who had no history of testicular trauma. We found an unexpectedly high incidence of significant testicular trauma in adolescence and young adulthood and hormonal differences between posttrauma patients, other infertile men, and normal men that may affect spermatogenesis.
MATERIALS AND METHODS
The study protocol was approved by the Committee for the Protection of Human Subjects of the University of Wisconsin. Participants gave informed consent after being provided with a full explanation of the purpose and extent ofthis investigation. Experimental Subjects and Study Design
One hundred seventy-nine infertile men, referred by gynecologists who had performed a preliminary investigation of the female partner, were evaluated at the Andrology Clinic of the University ofWisconsin. Infertile men were defined as men from couples with primary or secondary infertility, with ~1 year of attempted conception, who presented with abnormal parameters upon repeated semen analyses. A detailed history was obtained by questionnaire and personal interview. Inquiries were directed to the patient's lifestyle, the use of medications or recreational drugs, and details of sexual practice. Special attention was given to the time and circumstances of possible testicular trauma, including details regarding the intensity and duration of pain, the occurrence of post-traumatic swelling and scrotal hematoma, and the therapeutic measures taken, if any. To be considered significant, testicular trauma had to be followed by pain for ~24 hours and be associated with significant swelling and/or hematoma. The patients were examined for sexspecific body build and hair distribution, gynecomastia, manifestations of hyperthyroidism, and possible testicular atrophy. Total testicular volume was determined by comparing the size of each testicle to that of orchidometer beads. Patients with Klinefelter's syndrome, liver disease, renal failure, hyperthyroidism, or infectious orchitis, those who abused illicit drugs or alcohol, who used androgenic steroids, estrogens, or antiestrogens, or ingested spironolactone were excluded from the study. None 144
Nolten et al.
Testicular trauma and E2
of the study participants had a history of infrequent intercourse during the preceding year or had used personal lubricants. Patients whose partners had a significant infertility factor were not included. Forty-six healthy, age-matched men, who had recently fathered a child and denied a history oftesticular trauma, served as controls. Information about their past medical history and lifestyle was obtained by questionnaire and the same exclusion criteria recorded above were used. All study participants provided venous blood samples in the fasting state, between 8:00 and 10:00 A.M. Serum was separated by centrifugation after the drawing and stored at -70°C for later assays. Ejaculates were obtained in the clinic by masturbation, after 48 to 72 hours of sexual abstinence, and were evaluated immediately after liquefication. Materials and Assays
Follicle-stimulating hormone and LH were measured by a 1251 double antibody RIA, using kits from Diagnostic Products Corporation (Los Angeles, CA). The intra-assay and interassay coefficients of variation (CVs) of replicate samples were 3.2% and 4.8% for FSH and 2.9% and 5.3% for LH, respectively. The male reference range for FSH and LH is 1 to 15 mlU/mL (1 to 15 IU/L). Prolactin was measured by a reverse sandwichtype RIA, using a kit from Corning Medical (Medfield, MA). The intra-assay and interassay CVs of replicate samples were 3.2% and 5.1 %, respectively. The male reference range is 2 to 12 ng/mL (2 to 12 p,g/L). Testosterone was measured using a kit from ICN Biomedicals, Inc. (Costa Mesa, CA). The intra-assay and interassay CV s of replicate samples were 3.3% and 4.3%, respectively. The reference range for normal men is 400 to 1,000 ng/dL (13.9 to 34.7 nmoljL). The assay for bioavailable T was based on a modification of the method by O'Conner et al. (3). The percent unbound, bioavailable T was determined by equilibration of a serum aliquot with 2,000 cpm of 3H-T and precipitation of the sex hormone-binding globulin (SHBG)-bound T with ammonium sulfate at a final concentration of 50%. Serum preheated to 60°C for 30 minutes, to destroy SHBG, served as assay control. Multiplication of percent bioavailable T by total T yielded the bioavailable T concentration. The intra -assay and interassay CVs of rep Ii cate samples of low and high controls were 1.8% and 2.6% and 2.9% and 4.8%, respectively. The Fertility and Sterility
reference range for men is 67 to 400 ngjdL (2.3 to 13.9 nmoljL). Estradiol was measured after extraction of serum with ethyl acetate:hexane (3:2), using a kit from Pantex (Santa Monica, CA). The intra-assay and interassay CVs of replicate samples were 3.2% and 4.6%, respectively. The male reference range is 8 to 35 pgjmL (29.4 to 128.5 pmoljL). Dehydroepiandrosterone sulfate was measured by RIA, using a kit from Diagnostic Products Corporation. The intra-assay and interassay CVs of replicate samples were 3.1 % and 5.4 %, respectively. The reference range is 80 to 560 ~gjdL (2.2 to 15.2 ~moljL).
17 -Hydroxyprogesterone was determined by RIA, using a kit from Diagnostic Products Corporation. The intra-assay and interassay CVs of replicate samples were 4.7% and 5.1%, respectively. The reference range is 0.2 to 2.0 ngjmL (0.6 to 6.0 nmoljL). Sex hormone-binding globulin was measured with a sandwich-type noncompetitive "liquid phase" RIA kit obtained from Nuclear Diagnostics Inc. (Troy, MI). The intra-assay and interassay CVs of replicate samples were 3.8% and 5.3%, respectively. The reference range is 1.3 to 5.5 ~gjmL (13 to 55 nmoljL). The ejaculates were examined by automated computer-assisted semen analysis, using the Cellsoft System, Series 3000 (Cryo Resources, Montgomery, NY). Sperm morphology was determined manually. Measured parameters included ejaculate volume (normal, ~2.0 mL), total sperm count (normal, ~40 X 106 ), sperm motility (normal, ~50%), and percentage of abnormal sperm forms (normal, ~50%) (4). Data Analysis
Statistical methods used were regression analysis, analysis of variance, Student's t-test, X2, and descriptive statistics. Analysis was performed using the Statview II Professional Package (Abacus Concepts, Inc., Berkeley, CA) . Values are means ± SE.
RESULTS Of 179 infertile study participants, 30 (16.8%) reported at least one incident of significant blunt testicular trauma. The remaining 149 patients presented with infertility without history of testicular injury. As determined by the exclusion criteria, Vol. 62, No.1, July 1994
none of the 46 fertile men had experienced significant testicular injury. Clinical data and laboratory findings of men with testicular trauma were compared with those of infertile men without testicular trauma and of fertile men. Mean age of men with testicular trauma was 31.5 ± 9 years (men with testicular trauma versus infertile men without testicular trauma, P = 0.04), of infertile men without testicular trauma was 33.3 ± .4 years, and of fertile men was 33.7 ± .9 years. The mean body mass index (BMI = weight [kg]j height [m]2) in men with testicular trauma was 26.7 ± 1.0 (men with testicular trauma versus fertile men, P = 0.002), in infertile men without testicular trauma was 27.1 ±.5 (infertile men without testicular trauma versus fertile men, P = 0.0001), and in fertile men was 23.4 ± 0.4. Mean total testicular volume was 52 ± 3 mL in men with testicular trauma and 54 ± 1 mL in infertile men without testicular trauma. Testicular injury occurred at age ~1O years in five subjects, between ages 11 and 18 years in 21 patients, and above 18 years in four individuals. Mean age at injury was 15.1 ± 0.9 years; mean age at evaluation was 31.5 ± 0.9 years. The mean time interval between injury and evaluation was 16.4 ± 1.4 years. Sports-related causes of testicular trauma were playing football (n = 7), bicycling (n = 7), wrestling and martial arts (n = 5), baseball (n = 4), and basketball (n = 2). Five cases oftesticular injury unrelated to sports were due to automobile accidents, criminal assault, or unknown cause. Only one patient had seen a physician after the testicular injury. He had undergone evacuation of a scrotal hematoma and partial orchiectomy for testicular rupture. Self treatment of the other men with testicular trauma was limited to ingestion of analgesics and rest. Table 1 shows the results of semen analyses in the groups of study patients. Mean total sperm count and sperm motility were lower and percentage of abnormal sperm forms was higher in men with testicular trauma and in infertile men without testicular trauma than in fertile men. Statistically, the most significant difference in semen parameters in men with testicular trauma versus fertile men and in infertile men without testicular trauma versus fertile men was the decrease in sperm motility. Ejaculate volumes in all three groups and semen parameters in men with testicular trauma and in infertile men without testicular trauma were not significantly different from each other. Nolten et al.
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$' •
Table 1
Semen Analyses in Groups of Study Patients Men with testicular trauma (n = 30)
Subject group Ejaculate volume (mL) Total sperm count (X10 6 ) Sperm motility (%) Abnormal sperm forms (%)
2.8 154.0 41.8 22.2
± 0.3 ± 59.0 ± 4.0 ± 2.2
* Significantly different from men with testicular trauma = 0.02) and infertile men without testicular trauma (P = 0.002). t Significantly different from men with testicular trauma (P = 0.0001) and infertile men without testicular trauma (P = 0.0001).
Infertile men without testicular trauma (n = 149) 3.2 156.0 41.5 21.4
± 0.1 ± 18.0 ± 1.9 ± 0.9
Fertile men (n = 46) 4.2 290.0 62.6 16.5
± 0.9 ± 27.0* ± 2.5t ± 1.0*
* Significantly different from men with testicular trauma
(P
(P = 0.008) and infertile men without testicular trauma (P = 0.003).
Table 2 presents mean serum concentrations of reproductive hormones and SHBG in the groups of study patients. Estradiol in men with testicular trauma was at the upper limit of normal and 19% and 25% higher than in infertile men without testicular trauma and in fertile men, respectively. These differences in E2 concentrations were significant. Both FSH and LH were higher in men with testicular trauma and in infertile men without testicular trauma than in fertile men, with the highest values in men with testicular trauma. Mean FSH in men with testicular trauma was elevated above the reference range. There were no differences in PRL, T, percent bioavailable T, concentration of bioavailable T, and SHBG. Dehydroepiandrosterone sulfate and 17 -hydroxyprogesterone were likewise unchanged (data not shown). Serum concentrations of E2 and T were correlated positively in fertile men (P = 0.0002) and in infertile men without testicular trauma (P = 0.019). In contrast, there was no correlation ofE2 with T in men with testicular trauma (P = 0.79). Estradiol
was not correlated with FSH (P = 0.52; P = 0.15; P = 0.53), LH (P = 0.19; P = 0.62; P = 0.66), or BMI (P = 0.90; P = 0.60; P = 0.09) in fertile men, infer-
Table 2
DISCUSSION
In the majority of patients we are unable to identify the cause of male infertility. This study may help in defining the etiology of idiopathic infertility in some men, as it reports an unexpectedly high incidence of testicular trauma and associates testicular injury with permanent hormonal changes. A history of significant blunt remote testicular trauma that occurred with sports activities, accidents, or assault was reported by a high proportion
Reproductive Hormone and SHBG Concentrations in Groups of Study Patients Men with testicular trauma
Subject group FSH (mIU/mL)* LH (mIU/mL)* PRL (ng/mL)* E2 (pg/mL)1f T (ng/dL)tt Percent bioavailable T Bioavailable T (ngjdL)tt SHBG (/lg/mLlt*
17.9 12.0 6.7 35.0 558.0 26.9 152.0 2.2
± 3.8t ± 2.7§ ± 0.3 ± 1.7** ± 26.0 ± 2.2 ± 14.0 ± 0.3
* Conversion factor to SI units, 1.00. t Significantly different from fertile men (P = 0.002). * Significantly different from fertile men, P = 0.003. § Significantly different from fertile men, P = 0.005. II Significantly different from fertile men, P = 0.007.
146
tile men without testicular trauma, or men with testicular trauma. Similarly, we found no correlation between age at injury and either semen measurements or hormone levels in men with testicular trauma. Nor was there a difference in these parameters between subjects with prepubertal or postpubertal testicular injuries.
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Testicular trauma and E2
Infertile men without testicular trauma 13.9 8.7 6.3 29.5 516.0 27.2 139.0 1.9
± 1.2* ± 0.611 ± 0.2 ± 1.0 ± 11.0 ± 0.7 ± 5.0 ± 0.1
Fertile men 7.5 5.5 6.7 28.0 553.0 25.6 146.0 2.2
± 0.7 ± .3 ± 0.5 ± 1.3 ± 21.0 ± 1.5 ± 12.0 ± 0.2
1f Conversion factor to SI units, 3.671. ** Significantly different from infertile men without testicular trauma (P = 0.01) and fertile men (P = 0.001). tt Conversion factor to SI units, 3.467. H Conversion factor to SI units, 10.
Fertility and Sterility
of men who presented with infertility. Middle and high school students were the group at highest risk of testicular injury. Activities most commonly associated with testicular trauma were football, bicycling, and wrestling-martial arts. This distribution may relate to the relative popularity of these sports with young men from the area where the study was conducted. The incidence of blunt testicular trauma in infertile males has not been reported previously, and testicular injury is not commonly given consideration in the evaluation of male infertility. This could be explained by the fact that men who are seen for infertility often do not undergo the same careful, historical evaluation that is customary for other medical problems. Moreover, blunt testicular trauma apparently is often not considered as worthy of medical attention, unless it results in a surgical emergency. This is suggested by the observation that of 30 young men who experienced testicular trauma, only 1 sought and received medical assistance at the time of injury. Lack of regard for this problem results in lack of appreciation of its potential consequences. Men with testicular trauma and infertile men without testicular trauma had significantly lower sperm counts, lower sperm motility, and higher percentages of abnormal sperm forms than fertile men. The most significant difference was a decline in sperm motility. Reports on in vivo fertilization and IVF have shown that sperm count, motility, and percentage of normal forms correlate with fertility, and that both motility and morphology have predictive value for fertilization (5-7). There were no differences in testicular size or semen parameters between men with testicular trauma and infertile men without testicular trauma. As no other causes of male infertility were identified in men with testicular trauma, we only can conjecture that the observed seminal changes are the consequence of remote testicular trauma and that these abnormalities in semen parameters have a negative effect on fertility. Only a few recent publications discuss the effects of testicular trauma on semen parameters, reproductive hormones, and fertility. Koller et al. (1) followed six patients with a history of surgically treated unilateral testicular trauma for up to 8 years after injury and reported asthenospermia in all and abnormal sperm concentrations in most ejaculates, whereas serum concentrations of LH, FSH, T, E 2, and PRL remained within the normal range (1). Cass et al. (8), and Cass and Luxenberg Vol. 62, No.1, July 1994
(9) obtained semen analyses in cases of bilateral testicular injury after partial orchiectomy. The authors noted borderline semen analyses at 8 and 12 months after the accident. Daugaard and coworkers (2) examined 10 male torture victims, who had been subjected to genital trauma up to 5 years before evaluation by the Amnesty International Medical Group. The authors (2) found no significant changes in serum LH, FSH, T, and PRL in victims of genital torture when compared with a control group. In these publications, only small numbers of trauma patients were studied, and measurements in men with testicular injury were usually not compared with those in infertile men without history of testicular trauma and in agematched fertile controls, which makes interpretation of their results difficult. Slavis and co-workers (10) reported the effects of blunt unilateral testicular trauma on the fertility of adult Lewis rats. Whereas all normal males yielded offspring, only 27% of the traumatized animals were fertile. Even the contralateral testes showed decreased volumes, various degrees of spermatogenic arrest, and smaller seminiferous tubular diameters. These post-traumatic changes were explained by an autoimmune response of the contralateral gonad, after disruption of the blood-t~'~tis barrier of the injured testis. This animal study justifies the assumption that unilateral testicular injury may cause global testicular dysfunction with abnormal spermatogenesis and infertility. To investigate if remote testicular trauma causes permanent endocrine changes that might interfere with fertility, the serum concentrations of pituitary and testicular reproductive hormones and of SHBG were measured; whereas circulating levels of T, bioavailable T, and SHBG were similar in all subject groups, concentrations of LH and FSH were significantly higher in infertile men and highest in men with testicular trauma, with FSH being elevated above the reference range. Although differences between gonadotropin levels in men with testicular trauma and infertile men without testicular trauma were not significant, the trend to higher values in men with testicular trauma suggested more severe testicular dysfunction after trauma. Serum E2 concentrations were significantly higher in men with testicular trauma than in infertile men without testicular trauma and in fertile men; mean E2 in men with testicular trauma was at the upper limit of the reference range. This finding suggests that remote testicular trauma, suffered in childhood adolescence or early adulthood, may cause hormonal Nolten et ai.
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147
changes that can be documented in the fourth decade of life. Elevated E2 levels in men with testicular trauma could be caused by decreased metabolic clearance of E 2, by increased peripheral conversion of T or adrenal precursors to E 2, or by augmented testicular E2 secretion. In the absence of changes in SHBG, it can be assumed that the metabolic clearance of E2 in men with testicular trauma is identical to that in infertile men without testicular trauma or in fertile men. Peripheral blood levels of E2 reflect the sum of E2 derived from the peripheral conversion of precursors and of E2 secreted by the testes. Because there was no rise in circulating T, the testicular E2 precursor, it is unlikely that an increase in peripheral aromatization of T would cause increased E2 concentrations. In view of normal levels of 17 -hydroxyprogesterone, an adrenal precursor, and DHEAS, the derivative of an adrenal precursor ofE2, there is little to suggest that increased E2 production occurred from conversion of adrenal precursors through the 4-androstenedione-estrone pathway. The positive correlation of T with E2 in fertile men and in infertile men without testicular trauma shows that peripheral aromatization of T is a major source of E2 production in these subject groups. This is in agreement with findings by Hargreave et al. (11), who reported a positive correlation between E2 and T in infertile men. In contrast, there was no correlation of E2 with T in men with testicular trauma. This supports the assumption that increased E2 in men with testicular trauma does not originate from augmented peripheral aromatization of T, but rather from testicular E2 production. In normal men, 80% of circulating E2 is generated by peripheral aromatization ofT and only 20% originates from direct testicular secretion (12). Given the fact that most circulating E2 normally is generated by peripheral aromatization of T, and assuming that this contribution is consistent and that there is no change in peripheral metabolism of E 2, we conclude that, in men with testicular trauma, testicular E2 production must be increased markedly to raise E2 levels to values that are 25% above those measured in fertile men. Obese men have higher circulating E2 levels because of increased aromatization of androgens in adipose tissue, and a relationship between obesity, E2 levels, and infertility previously has been suggested (13, 14). In this study, BMI was slightly higher in men with testicular trauma and in infertile men without testicular trauma than in fertile 148
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Testicular trauma and E2
men; however, there was no correlation between E2 and BMI in any study group. If increased aromatization by adipose tissue were the cause of E2 elevation, the highest E2 levels might be expected in the group of study subjects with the highest BMI. As shown, E2 was not significantly higher in infertile men without testicular trauma, the group with the highest BMI, than in fertile men, the group with the lowest BMI. We conclude that the higher BMI is an unlikely cause ofE 2elevation in men with testicular trauma. We offer the following explanation for the posttraumatic increase in E2 concentration: after injury, the testicular mass decreases slightly and causes a compensatory rise of gonadotropins. Elevation of FSH will stimulate spermatogenesis and also testicular aromatase activity, which increases testicular E2 production and circulating E2 levels. The pertinent observation that FSH stimulates a cyclic AMP-dependent pathway that mediates E2 production in Sertoli cells was reported by Padmanabhan and co-workers (15) and by Rosselli and Skinner (16). Our supposition would be supported further by demonstration that higher E2 levels correlate with higher FSH concentrations in men with testicular trauma. However, such correlation was not noted, possibly because of the low number of study subjects and/or the episodic pattern of FSH secretion. Increased testicular E2 secretion may affect male fertility by the following mechanisms: E2 can decrease sperm production by inhibiting the synthesis oftesticular T, which is critical in spermatogenesis (17-19). Estradiol also can enhance catecholamine activity, produce ischemia of the seminiferous tubuli, and thus indirectly impair sperm production (20-23). Klaiber and Broverman (24) found increased E2 in smokers and a negative relationship between E2 levels and sperm counts, with smoking held constant. Reduction of E2 levels by blocking the conversion of T to E2 reportedly raises sperm counts in oligospermic men (25). We conclude that inhibition of spermatogenesis by high intratesticular E2 concentrations is a possible explanation for infertility in men with testicular trauma. Testicular trauma may result in formation of sperm antibodies that can impair fertility potential. However, such possible immunologic changes were not investigated. The importance of this preliminary study is in the identification of a subset of infertile men who have a common history of testicular trauma and, as a group, distinctive biochemical features. Although Fertility and Sterility
this initial study proposes an association between remote testicular trauma and male infertility, it neither proves it nor clearly identifies a specific pathophysiological mechanism. A prospective study focused on the early consequences of testicular injury with long-term follow-up will help to clarify the relationship of testicular trauma to infertility. The finding of a history of testicular trauma in a high percentage of infertile men suggests that greater attention should be paid both to the prevention of testicular injury and to its acute therapy. We suggest that adequate genital protective devices be used consistently by participants in contact sports to prevent testicular trauma that might have longterm consequences, including male infertility. Acknowledgments. The authors thank Ms. Peggy Nelson and Ms. Rachel Mikkelson of the Department of Medicine Secretarial Center for assistance in preparation of this manuscript. REFERENCES 1. Koller VA, Maier U, Pfliiger H. Das Hodentrauma und
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FERTILITY AND STERILITY Copyright
©
Printed on acid-free paper in U. S. A.
1994 The American Fertility Society
Association of elevated estradiol with remote testicular trauma in young infertile men
Wolfram E. Nolten, M.D.*t Sharon P. Viosca, B.S.:\: Stanley G. Korenman, M.D.:\:
Roya Mardi, B.A.:\: Sander S. Shapiro, M.D.§
University of Wisconsin, Madison, Wisconsin, and University of California Los Angeles (UCLA) Medical Center, Los Angeles, California
Objective: To determine the incidence of remote testicular trauma and of possible related permanent hormonal and seminal changes in infertile men. Design: Retrospective clinical study of hormonal and seminal parameters in a subpopulation of infertile men. Setting: Andrology Clinic in an academic research environment. Participants: Infertile men, with and without history of remote testicular trauma, and fertile volunteers. Main Outcome Measures: Percentage of infertile men with history of blunt testicular trauma, concentrations of reproductive hormones, and semen parameters. Results: Significant remote blunt testicular trauma was reported by 16.8% of infertile men. This had occurred 2 to 17 years (mean, 16.4 years) before evaluation, mostly with contact sports at adolescent age. Estradiol concentrations after testicular trauma were 19% and 25% higher than in infertile men without history of testicular injury and in fertile controls. Elevated E2 levels did not correlate with T. Infertile men with and without history of testicular trauma showed changes in seminal parameters. Conclusion: The incidence of remote blunt testicular trauma in infertile men is unexpectedly high. After injury, FSH-stimulated aromatization ofT may increase testicular E2 production, interfere with spermatogenesis, and cause infertility. Consistent use of protective devices in contact sports is recommended. Fertil Steril 1994;62:143-9 Key Words: Testicular trauma, E 2, male infertility
Blunt testicular trauma is not considered as a common cause of impaired spermatogenesis and altered testicular endocrine function (1, 2). Longterm hormonal consequences of testicular trauma have not been reported so far. Adolescent boys encounter a significant risk of testicular injury when Received August 30, 1993; revised and accepted March 9, 1994. * Reprint requests: Wolfram E. Nolten, M.D., Department of Medicine, University of Wisconsin Hospital and Clinics, H4/ 564 CSC, 600 Highland Avenue, Madison, Wisconsin 537925148 (FAX: 608-262-9289). t Department of Medicine, University of Wisconsin. :j: Department of Medicine, UCLA Medical Center. § Department of Obstetrics and Gynecology, University of Wisconsin. Vol. 62, No.1, July 1994
participating in contact sports. Protective cups enclose the testes but are often not used, and for many athletic activities no protection is required. Demonstration of permanent changes in testicular function caused by testicular trauma would support the need for better and more consistent protection of the young athlete. The evaluation of a large population of young adult men for suspected infertility offered an opportunity to study the prevalence of past testicular trauma in this group and to investigate the possibility that abnormalities in testicular function in adult life could be related to remote genital injury. To assess this possibility, we inquired with all male infertility patients about a possible history oftesticular trauma. Semen parameters and reproductive Nolten et al.
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hormone concentrations in infertile males who had experienced testicular trauma in the past were compared with those in infertile men without a history of testicular injury and with those in fertile men who had no history of testicular trauma. We found an unexpectedly high incidence of significant testicular trauma in adolescence and young adulthood and hormonal differences between posttrauma patients, other infertile men, and normal men that may affect spermatogenesis.
MATERIALS AND METHODS
The study protocol was approved by the Committee for the Protection of Human Subjects of the University of Wisconsin. Participants gave informed consent after being provided with a full explanation of the purpose and extent ofthis investigation. Experimental Subjects and Study Design
One hundred seventy-nine infertile men, referred by gynecologists who had performed a preliminary investigation of the female partner, were evaluated at the Andrology Clinic of the University ofWisconsin. Infertile men were defined as men from couples with primary or secondary infertility, with ~1 year of attempted conception, who presented with abnormal parameters upon repeated semen analyses. A detailed history was obtained by questionnaire and personal interview. Inquiries were directed to the patient's lifestyle, the use of medications or recreational drugs, and details of sexual practice. Special attention was given to the time and circumstances of possible testicular trauma, including details regarding the intensity and duration of pain, the occurrence of post-traumatic swelling and scrotal hematoma, and the therapeutic measures taken, if any. To be considered significant, testicular trauma had to be followed by pain for ~24 hours and be associated with significant swelling and/or hematoma. The patients were examined for sexspecific body build and hair distribution, gynecomastia, manifestations of hyperthyroidism, and possible testicular atrophy. Total testicular volume was determined by comparing the size of each testicle to that of orchidometer beads. Patients with Klinefelter's syndrome, liver disease, renal failure, hyperthyroidism, or infectious orchitis, those who abused illicit drugs or alcohol, who used androgenic steroids, estrogens, or antiestrogens, or ingested spironolactone were excluded from the study. None 144
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Testicular trauma and E2
of the study participants had a history of infrequent intercourse during the preceding year or had used personal lubricants. Patients whose partners had a significant infertility factor were not included. Forty-six healthy, age-matched men, who had recently fathered a child and denied a history oftesticular trauma, served as controls. Information about their past medical history and lifestyle was obtained by questionnaire and the same exclusion criteria recorded above were used. All study participants provided venous blood samples in the fasting state, between 8:00 and 10:00 A.M. Serum was separated by centrifugation after the drawing and stored at -70°C for later assays. Ejaculates were obtained in the clinic by masturbation, after 48 to 72 hours of sexual abstinence, and were evaluated immediately after liquefication. Materials and Assays
Follicle-stimulating hormone and LH were measured by a 1251 double antibody RIA, using kits from Diagnostic Products Corporation (Los Angeles, CA). The intra-assay and interassay coefficients of variation (CVs) of replicate samples were 3.2% and 4.8% for FSH and 2.9% and 5.3% for LH, respectively. The male reference range for FSH and LH is 1 to 15 mlU/mL (1 to 15 IU/L). Prolactin was measured by a reverse sandwichtype RIA, using a kit from Corning Medical (Medfield, MA). The intra-assay and interassay CVs of replicate samples were 3.2% and 5.1 %, respectively. The male reference range is 2 to 12 ng/mL (2 to 12 p,g/L). Testosterone was measured using a kit from ICN Biomedicals, Inc. (Costa Mesa, CA). The intra-assay and interassay CV s of replicate samples were 3.3% and 4.3%, respectively. The reference range for normal men is 400 to 1,000 ng/dL (13.9 to 34.7 nmoljL). The assay for bioavailable T was based on a modification of the method by O'Conner et al. (3). The percent unbound, bioavailable T was determined by equilibration of a serum aliquot with 2,000 cpm of 3H-T and precipitation of the sex hormone-binding globulin (SHBG)-bound T with ammonium sulfate at a final concentration of 50%. Serum preheated to 60°C for 30 minutes, to destroy SHBG, served as assay control. Multiplication of percent bioavailable T by total T yielded the bioavailable T concentration. The intra -assay and interassay CVs of rep Ii cate samples of low and high controls were 1.8% and 2.6% and 2.9% and 4.8%, respectively. The Fertility and Sterility
reference range for men is 67 to 400 ngjdL (2.3 to 13.9 nmoljL). Estradiol was measured after extraction of serum with ethyl acetate:hexane (3:2), using a kit from Pantex (Santa Monica, CA). The intra-assay and interassay CVs of replicate samples were 3.2% and 4.6%, respectively. The male reference range is 8 to 35 pgjmL (29.4 to 128.5 pmoljL). Dehydroepiandrosterone sulfate was measured by RIA, using a kit from Diagnostic Products Corporation. The intra-assay and interassay CVs of replicate samples were 3.1 % and 5.4 %, respectively. The reference range is 80 to 560 ~gjdL (2.2 to 15.2 ~moljL).
17 -Hydroxyprogesterone was determined by RIA, using a kit from Diagnostic Products Corporation. The intra-assay and interassay CVs of replicate samples were 4.7% and 5.1%, respectively. The reference range is 0.2 to 2.0 ngjmL (0.6 to 6.0 nmoljL). Sex hormone-binding globulin was measured with a sandwich-type noncompetitive "liquid phase" RIA kit obtained from Nuclear Diagnostics Inc. (Troy, MI). The intra-assay and interassay CVs of replicate samples were 3.8% and 5.3%, respectively. The reference range is 1.3 to 5.5 ~gjmL (13 to 55 nmoljL). The ejaculates were examined by automated computer-assisted semen analysis, using the Cellsoft System, Series 3000 (Cryo Resources, Montgomery, NY). Sperm morphology was determined manually. Measured parameters included ejaculate volume (normal, ~2.0 mL), total sperm count (normal, ~40 X 106 ), sperm motility (normal, ~50%), and percentage of abnormal sperm forms (normal, ~50%) (4). Data Analysis
Statistical methods used were regression analysis, analysis of variance, Student's t-test, X2, and descriptive statistics. Analysis was performed using the Statview II Professional Package (Abacus Concepts, Inc., Berkeley, CA) . Values are means ± SE.
RESULTS Of 179 infertile study participants, 30 (16.8%) reported at least one incident of significant blunt testicular trauma. The remaining 149 patients presented with infertility without history of testicular injury. As determined by the exclusion criteria, Vol. 62, No.1, July 1994
none of the 46 fertile men had experienced significant testicular injury. Clinical data and laboratory findings of men with testicular trauma were compared with those of infertile men without testicular trauma and of fertile men. Mean age of men with testicular trauma was 31.5 ± 9 years (men with testicular trauma versus infertile men without testicular trauma, P = 0.04), of infertile men without testicular trauma was 33.3 ± .4 years, and of fertile men was 33.7 ± .9 years. The mean body mass index (BMI = weight [kg]j height [m]2) in men with testicular trauma was 26.7 ± 1.0 (men with testicular trauma versus fertile men, P = 0.002), in infertile men without testicular trauma was 27.1 ±.5 (infertile men without testicular trauma versus fertile men, P = 0.0001), and in fertile men was 23.4 ± 0.4. Mean total testicular volume was 52 ± 3 mL in men with testicular trauma and 54 ± 1 mL in infertile men without testicular trauma. Testicular injury occurred at age ~1O years in five subjects, between ages 11 and 18 years in 21 patients, and above 18 years in four individuals. Mean age at injury was 15.1 ± 0.9 years; mean age at evaluation was 31.5 ± 0.9 years. The mean time interval between injury and evaluation was 16.4 ± 1.4 years. Sports-related causes of testicular trauma were playing football (n = 7), bicycling (n = 7), wrestling and martial arts (n = 5), baseball (n = 4), and basketball (n = 2). Five cases oftesticular injury unrelated to sports were due to automobile accidents, criminal assault, or unknown cause. Only one patient had seen a physician after the testicular injury. He had undergone evacuation of a scrotal hematoma and partial orchiectomy for testicular rupture. Self treatment of the other men with testicular trauma was limited to ingestion of analgesics and rest. Table 1 shows the results of semen analyses in the groups of study patients. Mean total sperm count and sperm motility were lower and percentage of abnormal sperm forms was higher in men with testicular trauma and in infertile men without testicular trauma than in fertile men. Statistically, the most significant difference in semen parameters in men with testicular trauma versus fertile men and in infertile men without testicular trauma versus fertile men was the decrease in sperm motility. Ejaculate volumes in all three groups and semen parameters in men with testicular trauma and in infertile men without testicular trauma were not significantly different from each other. Nolten et al.
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$' •
Table 1
Semen Analyses in Groups of Study Patients Men with testicular trauma (n = 30)
Subject group Ejaculate volume (mL) Total sperm count (X10 6 ) Sperm motility (%) Abnormal sperm forms (%)
2.8 154.0 41.8 22.2
± 0.3 ± 59.0 ± 4.0 ± 2.2
* Significantly different from men with testicular trauma = 0.02) and infertile men without testicular trauma (P = 0.002). t Significantly different from men with testicular trauma (P = 0.0001) and infertile men without testicular trauma (P = 0.0001).
Infertile men without testicular trauma (n = 149) 3.2 156.0 41.5 21.4
± 0.1 ± 18.0 ± 1.9 ± 0.9
Fertile men (n = 46) 4.2 290.0 62.6 16.5
± 0.9 ± 27.0* ± 2.5t ± 1.0*
* Significantly different from men with testicular trauma
(P
(P = 0.008) and infertile men without testicular trauma (P = 0.003).
Table 2 presents mean serum concentrations of reproductive hormones and SHBG in the groups of study patients. Estradiol in men with testicular trauma was at the upper limit of normal and 19% and 25% higher than in infertile men without testicular trauma and in fertile men, respectively. These differences in E2 concentrations were significant. Both FSH and LH were higher in men with testicular trauma and in infertile men without testicular trauma than in fertile men, with the highest values in men with testicular trauma. Mean FSH in men with testicular trauma was elevated above the reference range. There were no differences in PRL, T, percent bioavailable T, concentration of bioavailable T, and SHBG. Dehydroepiandrosterone sulfate and 17 -hydroxyprogesterone were likewise unchanged (data not shown). Serum concentrations of E2 and T were correlated positively in fertile men (P = 0.0002) and in infertile men without testicular trauma (P = 0.019). In contrast, there was no correlation ofE2 with T in men with testicular trauma (P = 0.79). Estradiol
was not correlated with FSH (P = 0.52; P = 0.15; P = 0.53), LH (P = 0.19; P = 0.62; P = 0.66), or BMI (P = 0.90; P = 0.60; P = 0.09) in fertile men, infer-
Table 2
DISCUSSION
In the majority of patients we are unable to identify the cause of male infertility. This study may help in defining the etiology of idiopathic infertility in some men, as it reports an unexpectedly high incidence of testicular trauma and associates testicular injury with permanent hormonal changes. A history of significant blunt remote testicular trauma that occurred with sports activities, accidents, or assault was reported by a high proportion
Reproductive Hormone and SHBG Concentrations in Groups of Study Patients Men with testicular trauma
Subject group FSH (mIU/mL)* LH (mIU/mL)* PRL (ng/mL)* E2 (pg/mL)1f T (ng/dL)tt Percent bioavailable T Bioavailable T (ngjdL)tt SHBG (/lg/mLlt*
17.9 12.0 6.7 35.0 558.0 26.9 152.0 2.2
± 3.8t ± 2.7§ ± 0.3 ± 1.7** ± 26.0 ± 2.2 ± 14.0 ± 0.3
* Conversion factor to SI units, 1.00. t Significantly different from fertile men (P = 0.002). * Significantly different from fertile men, P = 0.003. § Significantly different from fertile men, P = 0.005. II Significantly different from fertile men, P = 0.007.
146
tile men without testicular trauma, or men with testicular trauma. Similarly, we found no correlation between age at injury and either semen measurements or hormone levels in men with testicular trauma. Nor was there a difference in these parameters between subjects with prepubertal or postpubertal testicular injuries.
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Infertile men without testicular trauma 13.9 8.7 6.3 29.5 516.0 27.2 139.0 1.9
± 1.2* ± 0.611 ± 0.2 ± 1.0 ± 11.0 ± 0.7 ± 5.0 ± 0.1
Fertile men 7.5 5.5 6.7 28.0 553.0 25.6 146.0 2.2
± 0.7 ± .3 ± 0.5 ± 1.3 ± 21.0 ± 1.5 ± 12.0 ± 0.2
1f Conversion factor to SI units, 3.671. ** Significantly different from infertile men without testicular trauma (P = 0.01) and fertile men (P = 0.001). tt Conversion factor to SI units, 3.467. H Conversion factor to SI units, 10.
Fertility and Sterility
of men who presented with infertility. Middle and high school students were the group at highest risk of testicular injury. Activities most commonly associated with testicular trauma were football, bicycling, and wrestling-martial arts. This distribution may relate to the relative popularity of these sports with young men from the area where the study was conducted. The incidence of blunt testicular trauma in infertile males has not been reported previously, and testicular injury is not commonly given consideration in the evaluation of male infertility. This could be explained by the fact that men who are seen for infertility often do not undergo the same careful, historical evaluation that is customary for other medical problems. Moreover, blunt testicular trauma apparently is often not considered as worthy of medical attention, unless it results in a surgical emergency. This is suggested by the observation that of 30 young men who experienced testicular trauma, only 1 sought and received medical assistance at the time of injury. Lack of regard for this problem results in lack of appreciation of its potential consequences. Men with testicular trauma and infertile men without testicular trauma had significantly lower sperm counts, lower sperm motility, and higher percentages of abnormal sperm forms than fertile men. The most significant difference was a decline in sperm motility. Reports on in vivo fertilization and IVF have shown that sperm count, motility, and percentage of normal forms correlate with fertility, and that both motility and morphology have predictive value for fertilization (5-7). There were no differences in testicular size or semen parameters between men with testicular trauma and infertile men without testicular trauma. As no other causes of male infertility were identified in men with testicular trauma, we only can conjecture that the observed seminal changes are the consequence of remote testicular trauma and that these abnormalities in semen parameters have a negative effect on fertility. Only a few recent publications discuss the effects of testicular trauma on semen parameters, reproductive hormones, and fertility. Koller et al. (1) followed six patients with a history of surgically treated unilateral testicular trauma for up to 8 years after injury and reported asthenospermia in all and abnormal sperm concentrations in most ejaculates, whereas serum concentrations of LH, FSH, T, E 2, and PRL remained within the normal range (1). Cass et al. (8), and Cass and Luxenberg Vol. 62, No.1, July 1994
(9) obtained semen analyses in cases of bilateral testicular injury after partial orchiectomy. The authors noted borderline semen analyses at 8 and 12 months after the accident. Daugaard and coworkers (2) examined 10 male torture victims, who had been subjected to genital trauma up to 5 years before evaluation by the Amnesty International Medical Group. The authors (2) found no significant changes in serum LH, FSH, T, and PRL in victims of genital torture when compared with a control group. In these publications, only small numbers of trauma patients were studied, and measurements in men with testicular injury were usually not compared with those in infertile men without history of testicular trauma and in agematched fertile controls, which makes interpretation of their results difficult. Slavis and co-workers (10) reported the effects of blunt unilateral testicular trauma on the fertility of adult Lewis rats. Whereas all normal males yielded offspring, only 27% of the traumatized animals were fertile. Even the contralateral testes showed decreased volumes, various degrees of spermatogenic arrest, and smaller seminiferous tubular diameters. These post-traumatic changes were explained by an autoimmune response of the contralateral gonad, after disruption of the blood-t~'~tis barrier of the injured testis. This animal study justifies the assumption that unilateral testicular injury may cause global testicular dysfunction with abnormal spermatogenesis and infertility. To investigate if remote testicular trauma causes permanent endocrine changes that might interfere with fertility, the serum concentrations of pituitary and testicular reproductive hormones and of SHBG were measured; whereas circulating levels of T, bioavailable T, and SHBG were similar in all subject groups, concentrations of LH and FSH were significantly higher in infertile men and highest in men with testicular trauma, with FSH being elevated above the reference range. Although differences between gonadotropin levels in men with testicular trauma and infertile men without testicular trauma were not significant, the trend to higher values in men with testicular trauma suggested more severe testicular dysfunction after trauma. Serum E2 concentrations were significantly higher in men with testicular trauma than in infertile men without testicular trauma and in fertile men; mean E2 in men with testicular trauma was at the upper limit of the reference range. This finding suggests that remote testicular trauma, suffered in childhood adolescence or early adulthood, may cause hormonal Nolten et ai.
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147
changes that can be documented in the fourth decade of life. Elevated E2 levels in men with testicular trauma could be caused by decreased metabolic clearance of E 2, by increased peripheral conversion of T or adrenal precursors to E 2, or by augmented testicular E2 secretion. In the absence of changes in SHBG, it can be assumed that the metabolic clearance of E2 in men with testicular trauma is identical to that in infertile men without testicular trauma or in fertile men. Peripheral blood levels of E2 reflect the sum of E2 derived from the peripheral conversion of precursors and of E2 secreted by the testes. Because there was no rise in circulating T, the testicular E2 precursor, it is unlikely that an increase in peripheral aromatization of T would cause increased E2 concentrations. In view of normal levels of 17 -hydroxyprogesterone, an adrenal precursor, and DHEAS, the derivative of an adrenal precursor ofE2, there is little to suggest that increased E2 production occurred from conversion of adrenal precursors through the 4-androstenedione-estrone pathway. The positive correlation of T with E2 in fertile men and in infertile men without testicular trauma shows that peripheral aromatization of T is a major source of E2 production in these subject groups. This is in agreement with findings by Hargreave et al. (11), who reported a positive correlation between E2 and T in infertile men. In contrast, there was no correlation of E2 with T in men with testicular trauma. This supports the assumption that increased E2 in men with testicular trauma does not originate from augmented peripheral aromatization of T, but rather from testicular E2 production. In normal men, 80% of circulating E2 is generated by peripheral aromatization ofT and only 20% originates from direct testicular secretion (12). Given the fact that most circulating E2 normally is generated by peripheral aromatization of T, and assuming that this contribution is consistent and that there is no change in peripheral metabolism of E 2, we conclude that, in men with testicular trauma, testicular E2 production must be increased markedly to raise E2 levels to values that are 25% above those measured in fertile men. Obese men have higher circulating E2 levels because of increased aromatization of androgens in adipose tissue, and a relationship between obesity, E2 levels, and infertility previously has been suggested (13, 14). In this study, BMI was slightly higher in men with testicular trauma and in infertile men without testicular trauma than in fertile 148
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men; however, there was no correlation between E2 and BMI in any study group. If increased aromatization by adipose tissue were the cause of E2 elevation, the highest E2 levels might be expected in the group of study subjects with the highest BMI. As shown, E2 was not significantly higher in infertile men without testicular trauma, the group with the highest BMI, than in fertile men, the group with the lowest BMI. We conclude that the higher BMI is an unlikely cause ofE 2elevation in men with testicular trauma. We offer the following explanation for the posttraumatic increase in E2 concentration: after injury, the testicular mass decreases slightly and causes a compensatory rise of gonadotropins. Elevation of FSH will stimulate spermatogenesis and also testicular aromatase activity, which increases testicular E2 production and circulating E2 levels. The pertinent observation that FSH stimulates a cyclic AMP-dependent pathway that mediates E2 production in Sertoli cells was reported by Padmanabhan and co-workers (15) and by Rosselli and Skinner (16). Our supposition would be supported further by demonstration that higher E2 levels correlate with higher FSH concentrations in men with testicular trauma. However, such correlation was not noted, possibly because of the low number of study subjects and/or the episodic pattern of FSH secretion. Increased testicular E2 secretion may affect male fertility by the following mechanisms: E2 can decrease sperm production by inhibiting the synthesis oftesticular T, which is critical in spermatogenesis (17-19). Estradiol also can enhance catecholamine activity, produce ischemia of the seminiferous tubuli, and thus indirectly impair sperm production (20-23). Klaiber and Broverman (24) found increased E2 in smokers and a negative relationship between E2 levels and sperm counts, with smoking held constant. Reduction of E2 levels by blocking the conversion of T to E2 reportedly raises sperm counts in oligospermic men (25). We conclude that inhibition of spermatogenesis by high intratesticular E2 concentrations is a possible explanation for infertility in men with testicular trauma. Testicular trauma may result in formation of sperm antibodies that can impair fertility potential. However, such possible immunologic changes were not investigated. The importance of this preliminary study is in the identification of a subset of infertile men who have a common history of testicular trauma and, as a group, distinctive biochemical features. Although Fertility and Sterility
this initial study proposes an association between remote testicular trauma and male infertility, it neither proves it nor clearly identifies a specific pathophysiological mechanism. A prospective study focused on the early consequences of testicular injury with long-term follow-up will help to clarify the relationship of testicular trauma to infertility. The finding of a history of testicular trauma in a high percentage of infertile men suggests that greater attention should be paid both to the prevention of testicular injury and to its acute therapy. We suggest that adequate genital protective devices be used consistently by participants in contact sports to prevent testicular trauma that might have longterm consequences, including male infertility. Acknowledgments. The authors thank Ms. Peggy Nelson and Ms. Rachel Mikkelson of the Department of Medicine Secretarial Center for assistance in preparation of this manuscript. REFERENCES 1. Koller VA, Maier U, Pfliiger H. Das Hodentrauma und
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