Testis volumes, semen quality, and hormonal patterns in adolescents with and without a varicocele

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1 FERTILITY AND STERILITY Copyright e 1991 The American Fertility Society Printed on acid-free paper in U.S.A. Testis volumes, semen quality, and hormonal patterns in adolescents with and without a varicocele Lucien C. F. Haans, M.D.*t Joop S. K Laven, M.D.*:j: Willem P. Th. M. Mali, M.D., Ph.D. Egbert R. te Velde, M.D., Ph.D.* Cees J. G. Wensing, D.V.M., Ph.D.:j: University Hospital Utrecht, Utrecht, The Netherlands Objective: To study the effects of varicocele on testicular function in adolescents. Design: A prospective controlled study in 88 randomly selected adolescents. Setting: All participants were referred to the fertility outpatient clinic of our University Hospital. Participants: All participants with a grade II varicocele (group 2) or a grade III varicocele (group 3) were selected at a district military medical council, whereas a similar group of healthy volunteers without a varicocele served as controls (group 3). Interventions: Testis volumes were measured using an orchiometer. Semen analysis was performed according to standard procedures, and serum hormone levels were determined using a radioimmunoassay. Main Outcome Measure(s): Testis volumes, semen quality, and hormonal parameters in adolescents with and without a varicocele were compared. Results: In group 1 (n = 21), the mean left testis volume (24.5 ml; 95% confidence interval [CI]: 22.8 to 26.2) was significantly (P < 0.05) different from group 2 (n = 15) (20.9 ml; 95% CI: 18.5 to 23.4) and group 3 (n = 52) (20.7 ml; 95% CI: 19.2 to 22.2) (P < 0.01) adolescents. In adolescents with a pronounced varicocele-associated left testicular growth failure, the total sperm number was reduced. However, sperm concentration, motility, and morphology were not altered. Luteinizing hormone, follicle-stimulating hormone, testosterone, and prolactine levels were all within the normal ranges in the three groups. Conclusions: Left testicular growth failure in adolescents with a varicocele is only associated with a decrease in total sperm number. Fertil Steril 56:731, 1991 During the last decades, much attention has been given to varicocele as a potential cause of testicular dysfunction and subsequent male infertility. The mean incidence of varicocele among the male population is reported to be 15%. 1 Varicocele is detected in between 19% and 41% of male partners of infertile couples with abnormal semen quality. 2 Less is known about varicoceles in adolescents. A few studies report an incidence ranging from Received October 9, 1990; revised and accepted June 6, * Department of Obstetrics and Gynecology. t Reprint requests: Lucien C. F. Haans, M.D., Department of Obstetrics and Gynecology, University Hospital Utrecht, P.O. Box , 3508 GA Utrecht, The Netherlands. :j: Department of Anatomy, School of Veterinary Medicine. Department of Radiology, University Hospital Utrecht. 12.4% up to 25.8%. 3-7 Varicocele usually becomes clinically evident during pubertal development. Apparently, physiological changes in testis volume and blood supply during puberty are responsible for varicocele formation. 2 In up to 77% of adolescent varicocele carriers, unilateral or bilateral testicular growth failure has been reported. 8 9 Two reports with small numbers of patients describe some morphological abnormalities in testicular biopsies taken from young varicocele patients including premature sloughing of germinal cells and degenerative changes in Sertoli cells However, in approximately 50% of the studied subjects left testicular histology was normal. The decrease in testis size and the abnormal histologic findings in young varicocele patients are similar to those reported in adult subfertile varico- Haans et al. Effects of varicocele in adolescents 731

2 cele patients. Lyon and associates 8 reported reversal of testicular growth failure in a 17 -year-old boy after correction of a varicocele. In another report, 20 pubertal boys or adolescents with grade II or III varicoceles and a volume loss of the left testis were described. Preoperatively, the average volume of the left testis was 70% from that of the right side. After varicocele correction, this volume increased significantly to 91% in 16 of 20 patientsp Based on this rather scant literature, repair of varicoceles in adolescents is recommended when left testicular growth failure is present. It should be stressed that none of these studies were either prospective or controlled. We are conducting a prospective controlled study in adolescents with and without varicoceles to investigate the effects of varicocele on testicular function. In the present study, the clinical findings as well as semen and hormonal parameters at intake are reported. Patients MATERIALS AND METHODS Between March 1987 and December 1988, 67 male adolescents between 17 and 20 years of age were evaluated because of varicocele at the Department of Obstetrics and Gynecology of the University Hospital Utrecht. They were selected during a routine physical examination at a district military medical council. All 17 -year-old Dutch males are obliged by law to be physically examined and psychologically tested at these medical councils to establish their ability to serve as a draftee in the national army. During the same period, a similar group consisting of 21 male adolescent healthy volunteers 17 to 21 years of age serving as controls were examined. In this way, a virtually unbiased sample was drawn from the adolescent population. A complete medical history was obtained from all participants, and a physical examination was performed by one of the first two authors (L.C.F.H. or J.S.E.L.). None of the subjects reported previous episodes of cryptorchidism, hydrocele, testicular trauma, or had undergone surgery of the urogenital tract. The diagnosis of varicocele was based on physical examination in the erect and supine position using Valsalva's maneuver. Varicoceles were graded according to Dubin and Amelar 13 into: small (grade I), only detected during Valsalva's maneuver; moderate (grade II), easily palpated without Valsalva maneu- ver; large (grade III), causing visible bulging of the scrotal skin. Testis volumes were measured using an orchiometer according to Takihara et al. 14 The orchiometer was fitted over the stretched anterior scrotal skin, excluding the head and body of the epididymis, while the volume was recorded. Semen Analysis Two semen samples were collected after a period of sexual abstinence of at least 3 days. Semen was obtained at the hospital by masturbation. The time interval between the two consecutive semen samples was never <7 days and did not exceed 3 months. Semen analysis was performed immediately after collection according to standard World Health Organization 15 procedures. Volume, ph, consistency, and appearance of the ejaculate were determined. Motility was assessed in a wet drop preparation and designated as grade a (rapid linear progressive motility), b, c, or d (immotile). Sperm concentration was determined using a hemocytometer. Analysis of the morphological characteristics was performed in a stained smear. Testing for antibody coating of spermatozoa was achieved using the mixed antiglobulin reaction (MAR) test. 16 Finally, a capillary tube test according to Kremer and Jager 17 was performed to investigate sperm serum penetration. Hormone Measurements Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and prolactin (PRL) levels were determined in serum samples taken between 8.00 A.M. and 9.00 A.M. during the first visit. Serum LH and FSH were measured using a commercially available double-antibody radioimmunoassay (RIA) technique (Mallinkrodt, Dietzenbach, Germany). Results were expressed in IU of the First International Reference Preparation (IRP) (68/40) of LH and the second IRP (78/549) of FSH. The interassay variation was 9.4% at 13 IU/L (n = 41) for LH and 10% at 8 IU/L (n = 64) for FSH. Serum T was measured using an RIA after extraction with diethylether, as described previously by van Landeghem et al. 18 The interassay variation was 5.5% at 2.8 nmol/l (n = 13) and 10.3% at 1.5 nmolfl (n = 15). Prolactin levels were determined in a similar way using a commercially available RIA technique (IRE-Medgendix, Fleurus, Belgium). The interassay variation of this kit was 12.1% at 0.17 IU/L (n =52) and 7.3% at 0.77 IU/L (n = 69). 732 Haans et al. Effects of varicocele in adolescents Fertility and Sterility

3 Statistical Analysis In general, parametric tests were used when data were distributed normally (Kolgomorov-Smirnov), whereas nonparametric tests (Mann-Whitney U test) were used when a normal distribution was not present. For the comparison of means of the three different groups ANOV A was used. The resulting F value, if significant, was further scrutinized using Duncan's test for multiple comparisons to determine which groups were different from each other. Cochran's and Bartlett's tests were used to determine equality of variance. Chi-square tests were performed on the semiquantitative data of the sperm serum penetration assay. Analysis of covariance and regression statistics were used to investigate the correlation between testis volume and semen quality in adolescents with and without varicocele. The statistical significance level was considered to be present if the P value was <0.05. Statistical analysis was performed using a statistical software package (SPSS [Superior Performing Software Systems] Inc., Chicago, IL). Physical Examination RESULTS On the basis of the results of the physical examination, the 88 participants could be distributed over three groups. The first, group 1, consisted of 21 young healthy volunteers between 17 and 21 years of age (mean ± 1.02 [SD]). Group 2 consisted of 15 adolescents all 18 years of age with a grade II left-sided varicocele. Group 3 consisted of 52 adolescents between 17 and 21 years of age (mean ± 0.44 [SD]) with grade III varicoceles. Ages in the three consecutive groups did not differ significantly. In 2 adolescents of group 3, a bilateral varicocele was found, whereas in all the other cases varicoceles were exclusively found on the left side. The material included adolescents from both middle class and working class as well as boys from urban and rural areas. Virtually all adolescents had been unaware of the presence of the varicocele, none of them was referred to our department for fertility disorders, and none of them complained about varicoceleassociated symptoms such as scrotal pain or discomfort. The results of the testis volume measurements are summarized in Table 1. The mean left testis volumes of group 2 (20.9 ml; 95% confidence interval [CI]: 18.5 to 23.4) and group 3 (20.7 ml; 95% CI: 19.2 to 22.2) adolescents were significantly smaller Table 1 Testis Volumes of Adolescents With and Without Varicocele" Group Controls (n = 21) Grade II varicocele (n = 15) Grade III varicocele (n = 52) Left 24.5 ± ± 4.5b 20.7 ± 5.3 Right 24.7 ± ± ± 4.8 Values are ml means± SD. b Left testis volume of adolescents with grade II varicoceles was significantly different from controls (P < 0.05). Left testis volume of adolescents with grade III varicoceles was significantly different from controls (P < 0.01). as compared with the left mean testis volumes of the control group (24.5 ml; 95% CI: 22.8 to 26.2). Although right testis volumes were smaller too in adolescents with a varicocele, this reduction was not significant. In 1 of 15 adolescents in group 2 (7.1 %) and 3 of 52 in group 3 (5.8%), testis consistency was found to be abnormal. In all of the controls, testis consistency was normal. Semen Analysis Statistical analysis was performed on the best of the two initial semen samples. The best sample was determined using the total motile sperm count, i.e., volume X concentration X % motile spermatozoa. The results of the semen analysis are summarized in Figure 1. Briefly, no statistically significant differences were observed between the three consecutive groups. One adolescent from group 2 was found to be azoospermic. The number of adolescents with oligozoospermic counts ( <20 X 10 6 /ml) in groups 1, 2, and 3 were 19% (4/21), 20% (3/15), and 13.4% (8/52), respectively. Asthenozoospermia ( <40% progressive motility) was encountered in 17.9% (6/52) in group 3, but in none (0%) of the controls and in group 2. Teratozoospermia ( <40% morphologically normal spermatozoa) was observed in 14.3% (3/21) of the controls, whereas these figures were 26.7% (4/15) and 17.3% (9/52) in groups 2 and 3, respectively. As far as the MAR test was concerned, no significant differences in coating antibodies could be found. Sperm -coating antibodies of some clinical importance could not be detected in any of the participants in this study. Although the mean semen parameters were within the normal range, semen qualities tended to be lower in the varicocele subjects. It is possible that such a tendency becomes more apparent when the left testis volume is also taken into account. By the use of Haans et al. Effects of varicocele in adolescents 733

4 Normal count Normal count o Subnormal count o Subnonnal count Nonnal count o Subnormal count ] IZO i X I :. 100.g.. - l... I! l. == ' 80..t :., ';! :, : j I.. ( , !.._ -.- oog 0 q,o : lo lo.. ZOt-. :. 4 ""' 0 i 0 : Controls Grade II Grade Ill Controls Grade II Grade III Controls Grade II Grade Ill varicocele varicocele varicocele varicocele varicocele varicocele Figure 1 Individual sperm counts, motility percentages, and percentages of morphologically normal spermatozoa in controls (group 1), grade II varicoceles (group 2), and grade III varicoceles (group 3). Each dot represents an individual count or percentage. Open and closed dots represent subnormal and normal counts or percentages. Horizontal bars indicate the group means. 0 regression statistics, this was evaluated for the relation between left testis volume with total sperm number, sperm concentration, sperm motility, and sperm morphology both for subjects with and without varicoceles. Only the relationship between left testis volume and total sperm number in adolescents with a varicocele appeared to be statistically significant (r = 0.26, P = 0.03) (Fig. 2). The identical intercepts ( ± [SE] and ± [SE] in varicocele subjects and controls, respectively) and slopes (8.13 ± 3.74 [SE] and 7.73 ± 9.46 [SE] in varicocele subjects and controls, respectively) of both lines suggest a similar relationship in both groups. Analysis of covariance, correcting either for the presence of a varicocele or for the left testis volume, confirms that it is the volume and not the presence of a varicocele that determines this relationship. It is also obvious that subjects with a varicocele tend to have smaller left testis volumes, which merely confirms the data in Table 1. No statistically significant correlation could be found when the relationship between left testis volume and sperm concentration and morphology was studied. The sperm serum penetration assay did not reveal significant differences between the three groups. Progressive motility in serum lasted as long as 48 hours in 20 of 21 (95.2%) healthy volunteers (group 1). In groups 2 and 3, these percentages were 85.7% and 82.7%, respectively (P = 0.37). Sperm penetration densities at 1 em (distance traveled in capillary tube) after 2 hours of incubation exceeded 100 in 71.4%, 57.1 %, and 53.8% in groups 1, 2, and 3, re- spectively (P = 0.53). Similar results could be obtained when sperm densities at 3 and 4.5 em were taken into account. These migration reduction data are within normal limits. Motility grades at 1, 3, and 4.5 em after 2 hours' incubation were also within the normal range and did not differ significantly between the three groups. All participants had progressive motility grades a and/or b. Hormone Measurements All serum hormone levels of LH, FSH, T, and PRL were within the normal range. No statistically significant differences were present between the three groups, except for LH and T. The mean serum LH levels were significantly higher in group 3 (6.61 ± 2.60 IU/L, [SD])(P = 0.041) as compared with group 1 (5.10 ± 2.57 IU/L [SD]). Similarly, mean serum T levels were significantly higher in groups 2 (23.25 ± 7.51 nmol/l [SD])(P = 0.037) and 3 (23.52 ± 6.43 nmol/l [SD])(P = 0.011) as compared with group 1 (19.22 ± 5.73 nmol/l [SD]). To investigate the tendency of higher LH and T levels in the varicocele subjects, regression statistics were used to study the relationship between the left testis volume and the different hormonal parameters and the LH/T ratio. Only in adolescents with a varicocele, the relationship between the left testis volume and the LH/T ratio appeared to be statistically significant (r = -0.34, P = 0.005). The identical intercepts (0.47 ± 0.06 [SE] and 0.40 ± 0.19 [SE] in varicocele and control subjects, respectively) and slopes ( ± [SE] and ± [SE] 734 Haans et al. Effects of varicocele in adolescents Fertility and Sterility

5 .li 800 l o.s I o.s 0.4 O.J O.l : ,o Left Testis Volume.. : \ \"' -- a =--"-i---! -- VAR j CON Left Testis Volume Figure 2 Scatter diagram showing the left testis volume and the total sperm number or the LH/T ratio for both adolescents with a varicocele (e) and controls (0) with the two regression lines. V AR represents the regression line of the varicocele subjects, whereas CON represents the regression line in the control group. in varicocele and control subjects, respectively) again suggest a similar relationship between the left testis volume and the LH/T ratio in both controls and varicocele subjects (Fig. 2). Analysis of covariance confirms that the left testis volume and not the presence of a varicocele determines the LH/T ratio. It appears that the subjects with the smallest left testis volume tend to have the highest LH/T ratio. No statistical significant correlation could be obtained when the relationship between the left testis volume and LH, FSH, T, and PRL was studied. DISCUSSION Left testicular growth failure was present in adolescents with moderate to large varicoceles; the noted right testicular growth failure was not statis- tically significant. The degree of the varicocele did not have a consistent relation to the left and right testis sizes. These findings are in agreement with those reported by others Several investigators have reported varicoceleassociated changes in the spermatogenic epithelium of the testis in varicocele patients These changes were especially pronounced in those patients with a marked reduction in testis size. In the present study, mean semen parameters in both adolescents with and without varicoceles were not different and all within normal ranges, although asthenozoospermic and teratozoospermic counts tended to be more common in varicocele subjects than in controls. However, in subjects with a pronounced reduction in testis size, mean semen parameters did not seem to be different from subjects with slight or no reduction of testis size at all except for the total sperm number. The latter parameter was positively correlated with the degree of left testicular growth failure. The spermatogenic epithelium in these adolescents with a varicocele, however, is functioning within normal ranges as indicated by the normal mean semen quality parameters. A possible explanation for these dissimilarities with the earlier mentioned reports could reside in the differences in age and pubertal development. Changes in testis volume are reflected in changes in growth and length of the seminiferous tubules and concomitantly in sperm output, i.e., the total sperm number. 20 It seems, however, also possible that varicocele-associated testicular growth failure, although yet subtle, could induce testicular dysfunction, leading to future fertility problems. Furthermore, if this appears to be true, the question still remains whether or not treatment will stop or even reverse testicular dysfunction. The further follow-up study may provide an answer to these questions. Although hormonal parameters were within the normal ranges in subjects with and without varicoceles, the serum LH and T levels tended to be higher in the varicocele groups. Moreover, when a pronounced testicular growth failure was present, levels of the LH/T ratio tended to be higher in varicocele subjects. These results are somewhat puzzling because slightly increased serum LH levels are usually combined with lowered T levels in varicocele patients Apparently, varicocele-associated testicular growth failure in adolescents is also associated with a change in Leydig cell responsiveness. At present, there is no uniformity in the guidelines for the management of adolescents with a varicocele. However, most investigators nowadays recommend Haans et al. Effects of varicocele in adolescents 735

6 early repair when varicocele is associated with a reduction in testis size These recommendations are mainly based on the findings that left testicular growth failure and histologic changes in adolescents are similar to those reported in the adult subfertile varicocele patient. In the present study, varicocele-associated testicular growth failure could not clearly be associated with a pronounced testicular dysfunction as reflected by decreased semen qualities. In the light of this finding and because the reported testicular growth failure as well as the histologic abnormalities associated with varicocele in boys cannot be definitely attributed to varicocele, the recommendations concerning early treatment are questionable. In our opinion, a close follow-up of pubertal boys and adolescents with varicoceles seems to be more appropriate. In conclusion, the present study indicates that varicocele-associated unilateral or bilateral testicular growth failure is not clearly associated with a decrease in semen quality. Furthermore, it is not clear whether or not this growth failure continues during adulthood and could lead to future disturbances in fertility. Acknowledgments. The authors thank all the personnel of the military medical councils as well as the medical staff of the Dutch Armed Forces, especially M. van Ormondt, M.D., Ministry of Defense, Kerkrade, The Netherlands. Furthermore, we are very grateful to all personnel of the fertility and endocrinology laboratories of the University Hospital Utrecht, Utrecht, The Netherlands. Finally, we thank J.M. Eimers, M.Sc., from the Department of Public Health and Social Medicine, Erasmus University, Rotterdam, The Netherlands, for her statistical assistance. REFERENCES 1. Saypol DC: Varicocele. J Androl2:61, Pryor JL, Howards SS: Varicocele. Urol Clin North Am 14: 499, Homer JS: The varicocele: a survey among secondary schoolboys. Med Officer 104:377, Oster J: Varicocele in children and adolescents: an investigation of the incidence among Danish school children. Scand J Urol Nephrol 5:27, Steeno 0, Knops J, Declerck L, Adimoelja A, van de Voorde H: Prevention of fertility disorders by detection and treatment of varicocele at school and college age. Andrologia 8:4 7, Y erokhin AP: Classification and frequency of varicocele in children. Klin Khir 6:45, Berger OB: Varicocele in adolescence. Clin Pediatr (Phila) 19:810, Lyon RP, Marshall S, Scott MP: Varicocele in childhood and adolescence: implication in adulthood infertility? Urology 19: 641, Pozza D, D'Ottavio G, Masci P: Left varicocele at puberty. Urology 22:271, Hienz HA, Voggenthaler J, Weissbach L: Histological findings in testes with varicocele during childhood and their therapeutic consequences. Eur J Pediatr 133:139, Kass EJ, Chandra RS, Belman AB: Testicular histology in the adolescent with a varicocele. Pediatrics 79:996, Kass EJ, Belman AB: Reversal of testicular growth failure by varicocele ligation. J Urol137:475, Dubin L, Amelar RD: Varicocele size and results of varicocelectomy in selected subfertile men with a varicocele. Fertil Steril 21:606, Takihara H, Sakatoku J, Fujii M, Nasu T, Cosentino MJ, Cockett ATK: Significance of testicular size measurements in andrology.l. A new orchiometer and its clinical application. Fertil Steril 39:836, World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Semen -Cervical Mucus Interaction, 2nd edition. Cambridge, The Press Syndicate of the University of Cambridge, 1987, p Jager S, Kremer J, Kuiken J, Slochteren van-draaisma T: Immunoglobulin class of antispermatozoal antibodies from infertile men and inhibition of in vitro sperm penetration into cervical mucucs. Int J Androl 3:1, Kremer J, Jager S: Characteristics of anti-spermatozoal antibodies responsible for the shaking phenomenon, with special regard to immunoglobulin class and antigen-reactive sites. Int J Androl 3:143, van Landeghem AAJ, Poortman J, Deshpande N, DiMartina L, Tarquini L, Thijssen JHH, Schwarz F: Plasmaconcentration gradient of steroid hormones across human mammary tumours. J Steroid Biochem 14:741, Lipshultz LI, Corriere JN: Progressive testicular atrophy in the varicocele patient. J Urol 117:175, Straaten HWM, Wensing CJG: Histomorphometric aspects of testicular morphogenesis in the pig. Bioi Reprod 17:467, Comhaire F: Study of the hypothalamo-pituitary-testicular function in patients with varicocele before and after operation. In Progress in Reproductive Biology: Sperm Action, Edited by PO Hubinont, M L'Hermite. Basel, Karger, 1976, p Hudson RW, McKay DE: The gonadotropin response of men with varicoceles to gonadotropin-releasing-hormone. Fertil Steril33:427, Haans et al. Effects of varicocele in adolescents Fertility and Sterility