Hernia DOI 10.1007/s10029-015-1361-x
ORIGINAL ARTICLE
Impact of varicocele on biological markers of gonadal function E. Blevrakis • E. Chatzidarellis • D. Anyfantakis G. Sakellaris • M. Raissaki • O. Zoras • C. Mamoulakis • F. Sofras • E. Chrysos
•
Received: 14 August 2014 / Accepted: 20 February 2015 Ó Springer-Verlag France 2015
Abstract Purpose The aim of this study is to evaluate the potential effect of varicocele in the hormonal and clinical profile of adolescents. Methods Twenty adolescents at Tanner stage 4–5 with left varicocele were studied and compared with a control group of 20 healthy adolescents. All patients underwent ultrasonographic testicular volumetry as well as hormonal evaluation of inhibin B, testosterone, baseline and gonadotropin-releasing hormone stimulated, follicle-stimulating hormone as well as luteinizing hormone.
Statistical analysis was performed using the student’s t test with p value \0.05 taken as statistical significant. Results Patients with varicocele showed reduced levels of inhibin B compared to controls and a significant reduction in the testicular volume on the affected side. The response of luteinizing hormone to gonadotropin-releasing hormone stimulation was significantly higher in the varicocele group compared to the control group. Furthermore a significant inverse relationship of inhibin B compared to follicle-stimulating hormone was noted. Conclusion Serum inhibin B levels could represent a useful marker of Sertoli cell damage caused by varicocele. Keywords
E. Blevrakis (&) G. Sakellaris Department of Pediatric Surgery, University Hospital of Heraklion, Crete, Greece e-mail: [email protected] E. Chatzidarellis Department of Urology, Iaso General Hospital, Athens, Greece D. Anyfantakis Primary Health Care Centre of Kissamos, Chania, Crete, Greece M. Raissaki Department of Radiology, University Hospital of Heraklion, Crete, Greece O. Zoras Department of Surgical Oncology, University Hospital of Heraklion, Crete, Greece C. Mamoulakis F. Sofras Department of Urology, University Hospital of Heraklion, Crete, Greece E. Chrysos Department of General Surgery, University Hospital of Heraklion, Crete, Greece
Inhibin B Varicocele Gonadal function
Introduction Varicocele represents the abnormal dilatation of the pampiniform venous plexus inside the spermatic cord affecting about 15 % of the general male population and up to 40 % of males presenting to infertility clinics [1]. The condition is also detected in 2–11 % of prepubertal males [2–4] with a reported increase to 15–16 % in postpubertal adolescent males [5]. Although specific therapy is required in only 2 out of 10 affected males, the condition frequently leads to male infertility [6]. Pathophysiological mechanism through which varicocele results to testicular dysfunction has not yet been completely clarified. Potential etiollogies, include hormonal alterations, increase of non-collagenous proteins and immunoglobulins in the spermatic vein [6], production of toxic substances [7] as well as testicular hypoxia [6]. With regard to hormonal changes, it is still unknown whether these are the cause of gonadal damage.
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Hernia
Previous research efforts have attempted to identify parameters of testicular dysfunction, such as gonadotropin levels before and after stimulation with GnRH [8]. Inhibin B serum levels have been investigated in a group of young patients with varicocele at different Tanner developmental stages [9]. The authors concluded that the hormonal profile in prepubertal and pubertal males with varicocele may be altered. This may suggest an early involvement in seminiferous epithelial function [9]. The aim of the present study was to evaluate the hormonal profile of adolescents with varicocele in an attempt to use biochemical markers for selection of patients requiring surgical treatment and prevent ongoing testicular damage.
Materials and methods Over a 3 years period, 20 adolescent males with left unilateral varicocele were included in the study. All patients were referred for evaluation of an asymptomatic varicocele detected during routine pediatric examination. Diagnosis was confirmed through physical examination by a paediatric surgeon. Varicoceles were clinically classified according to the classification of Dubin and Amelar [10]. Pubertal stage was defined using Tanner’s criteria [11]. Testicular volume was determined by ultrasonography. Testicular growth arrest was considered when the testicular volume of the affected testicle was smaller than 2 ml compared to the contralateral testicle. The control group consisted of 20 age matched healthy adolescents of similar pubertal development stages and free of any endocrinological disorder. Endocrine function tests were performed in both varicocele adolescents and controls including FSH, LH, testosterone and inhibin B serum levels as well as stimulated LH and FSH, 30 and 60 min after intravenous administration of 100 lg GnRH. Inhibin B, FSH, LH and testosterone were measured by standardized radioimmunoassay techniques using ELISA commercial kits. Serum from each patient and control
Table 1 Mean and SD of inhibin B, FSH, LH and testis volumes from varicocele group and controls
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Results All males had a left-sided varicocele. The mean age of the patients was 13.8 years (range 12–17) and the mean age of the control group was 14.5 years (range 12–17). The biochemical markers of varicocele and control groups are summarized in Table 1. Patients with varicocele showed a significant reduction in the testicular volume of the affected side (12.9 ± 2.33 ml on the left vs 15.9 ± 2.32 ml on the right; p = 0.0001) (Fig. 1), whereas no significant difference was observed in the testis volume of the control group 17.1 ± 2.36 ml on the left vs 16.1 ± 2.39 ml on the right). No statistically significant differences in basal FSH, LH, testosterone and the maximal response of FSH were found between subjects with varicocele and the control group. The response of LH to GnRH stimulation was significantly higher in the varicocele group compared to the control group (20.8 ± 6.86 vs 15.8 ± 3.65 mIU/mL, respectively; p \ 0.05) (Fig. 2). Inhibin B levels were significantly lower in the varicocele group when compared to the age-matched controls (337 ± 33.6 vs 384 ± 35 pg/mL, respectively; p \ 0.05) (Fig. 3). Inhibin B levels in varicocele patients were positively correlated with testicular volume (r = 0.8646; p \ 0.05). No significant correlation was observed between inhibin B and testosterone or both baseline and stimulated LH. However, the correlation coefficient of inhibin B compared with FSH was negative and statistical significant (r = -0.826; p \ 0.05).
Varicocele group
Control group
p
Inhibin B (pg/mL)
337 ± 33.6
384 ± 35
0.0001
Testosterone (ng/dL) FSH baseline (mIU/mL)
335 ± 242 3.95 ± 1.39
340 ± 21.6 3.1 ± 1.28
0.49 (NS) 0.051 (NS)
FSH peak (mIU/mL)
7.73 ± 3.14
6.25 ± 2.16
0.09 (NS)
3.9 ± 1.65
3.47 ± 1.45
0.39 (NS)
LH peak (mIU/mL)
20.8 ± 6.86
15.8 ± 3.65
0.0066
Testicular volume R (mL)
15.9 ± 2.32
16.1 ± 2.39
0.083 (NS)
Testicular volume L (mL)
12.9 ± 2.33
17.1 ± 2.36
0.0001
LH baseline (mIU/mL)
NS indicates not significant
adolescents was frozen at -80 °C until assayed. Informed consent was obtained by both study groups and their guardians. Data are presented as the mean ± SD. Statistical analysis was performed using the Student’s t test for paired observations, with p value \0.05 taken as statistical significant and by calculation of the Pearson’s-Correlation coefficient.
LEFT TESTICULAR VOLUME (ml)
Hernia
LEFT TESTICULAR VOLUME
30
CONTROL GROUP
20
VARICOCELE GROUP
10 0
0
10
20
30
PATIENTS
Fig. 1 A plot of left testes volume in the varicocele patients and in control group. (12.9 ± 2.33 ml on the left testes in the varicocele patients vs 17.1 ± 2.36 ml on the left in the control group)
INHIBIN
Fig. 2 The response of LH to GnRH stimulation was significantly higher in the varicocele group than those of the control group (20.8 ± 6.86 mIU/mL in the varicocele group vs 15.8 ± 3.65 mIU/ mL in the control group)
500 450 400 350 300 250 200 150 100 50 0
INHIBIN B
CONTROL GROUP VARICOCELE GROUP
0
5
10
15
20
25
PATIENTS
Fig. 3 Inhibin B levels were significantly lower in the varicocele group when compared with the age-matched controls (337 ± 33.6 vs 384 ± 35 pg/mL)
Discussion Inhibins are glycoproteins predominantly produced in the gonads [12]. Inhibin B, composed of a and b subunits, is the physiologically relevant form of inhibin in males [13]. Both subunits are produced by Sertoli cells, regulated by FSH and unidentified factors by germ cells [14]. Leydig cell products may also influence inhibin synthesis in Sertoli cells [15]. The serum profile of inhibins has been determined in normal males from birth to puberty. After infancy, inhibin B serum concentration falls within the lower range of adult normal levels. At approximately the age of 10 years old inhibin B serum concentration starts to rise,
concomitantly with testicular volume and serum testosterone, reaching adult levels at Tanner stage 2 [16]. Issues of reproductive health of men in the general population such as the semen quality has received been subject of research efforts [17]. However, obtainment of semen samples in adults is difficult. Instead blood samples are easier to obtain than ejaculates. Therefore, serum biomarkers of spermatogenesis are of major interest for population studies. Circulating FSH has previously been used as a marker of spermatogenesis—has long been considered a valuable marker for Sertoli cell function, but FSH is influenced by hypothalamic function, as well as by testicular factors and steroid hormones. Serum Inhibin B was recently suggested as a promising, more direct serum marker of Sertoli cell function and spermatogenesis than FSH [18]. Early gonadal abnomarlities in patients with varicocele have been ascribed mainly to the interstitial compartment of the testis. Ponchietti et al. [19] described many of the ultrastructural changes in the testis of the peri-pubertal adolescent with a varicocele. These changes include a reduced number and atrophy of the Sertoli cells, germ cells and Leydig cells. They also reported fibrotic changes in the testicle, as well as sclerosis of the capillaries within the testis. A concomitant impairment of seminiferous tubule function has also been documented [20]; however, little information is available on the possible role of the Sertoli cell as a primary site of injury in these patients. Measurement of testicular volume is considered important in evaluating the adolescent with a varicocele. A significant size discrepancy between the testicles has become the main indication for surgical treatment. Steeno et al. [21] first observed a loss of testicular volume and/or a change in the consistency of the testis ipsilateral to the varicocele, most often the left testis, in about a third of children with Grade 2 varicoceles and in 80 % of boys with grade 3 varicoceles. Lyon et al. [22] reported a volume loss of the left testis in 77 % of adolescent boys with a clearly palpable left varicocele. In our study, we have found a significant reduction in the volume of the affected left testis in the varicocele group compared with both the contralateral testis and the testis control. Our findings are in accordance with those of preoperative testicular volumetric asymmetry by these investigators, and support the view that a large adolescent varicocele can be responsible for testicular growth retardadion. Trigo et al. [12] found a significantly lower volume in the affected left testis in patients with varicocele at Tanner stage 3–4, but not during the first and second stages of puberty. This could be explained as progressive damage caused by the varicocele upon seminiferous tubule function during the latter stages of puberty. The management of varicocele in adolescents is still controversial. One of the controversial issues is the
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existence of prognostic markers that would make it possible to decide for surgery and to determine, when necessary, the appropriate time to perform it [23, 24]. In our opinion, a better knowledge of the natural history of the disease might help to resolve this dilemma. For this reason, in our study we have evaluated the different biochemical parameters in pubertal patients, and by correlating them, we have made an attempt to analyze their potential usefulness. However, it is worth mentioning that since our patient population could not be followed up until adulthood, it was not possible to find clear prognostic markers of testicular damage. Another study found that the evaluation of testicular volume is not predictive for testicular dysfunction in adolescence [25]. Moreover, they could not find any statistical difference in testosterone serum levels between varicocele patients without semen abnormalities [25]. They demonstrated a significant increase in basal LH, FSH, and FSH after GnRH stimulation in varicocele patients with semen dysfunction [25]. According to the authors, only FSH and LH dosage could help in identifying those subjects at risk for future fertility problems [25]. In alignment with our findings a study among 95 infertile varicocele patients found lower concentration levels of inhibin B in the peripheral blood and seminal plasma compared to a control group of fertile males [26]. On the contrary, other studies have that in pubertal stages 4 and 5 inhibin B was lower in the varicocele group than in controls [12, 27]. We have also found reduced inhibin B serum levels in males with varicocele compared with controls. All varicocele patients were at Tanner stages 4–5. In these patients, we observed a significant positive correlation between inhibin B and testis volume. There was no significant correlation (either positive or negative) between inhibin B and LH and testosterone. However, the correlation coefficient of inhibin B compared with FSH achieve a statistical relevance. Inhibin B, as we mentioned, is a glycoprotein produced by Sertoli Cells and regulated by FSH and other factors produced by germ cells [28]. Inhibin B secretion depends on the interaction between Sertoli cells and germ cells. In particular, it is known that spermatids influence inhibin production and that these cells are sensitive to hyperthermia [29]. It is possible that the elevated scrotal temperature associated with varicocele has impaired spermatid function, thus reducing inhibin B production [30]. Alternatively reduced inhibin B serum levels in the varicocele group could result from early arrest of spermatogenesis or a depletion of germ cells. This might be expected to be associated with reduced testicular volume, which we have observed in the varicocele affected testis associated with reduced inhibin B serum levels. It has been shown that there is an increased nitrotyrosine concentration within the spermatic vein on the affected side
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in varicocele patients. This would be consistent with our findings. Further studies are necessary to support this hypothesis. In conclusion, although reduced serum inhibin B levels; a significant inverse relationship of inhibin B compared with FSH and higher response of LH to GnRH stimulation measured in adolescent boys with varicocele, we were not able to identify other abnormalities. Given the diagnostic uncertainty of medical science [31, 32] and on the basis of literature data and these findings, inhibin B might be useful diagnostic marker of testicular damage caused by varicocele and assist in the selection of patients for surgical treatment to prevent infertility problems. The deterioration of the testicular function in adolescents with varicocele is a progressive phenomenon that requires an intensive follow up to identify early signs of testicular dysfunction and to prevent further testicular damage. Surgical repair of varicocele is the most appropriate therapy to avoid infertility [33]. Absolute indications for varicocelectomy in adolescents with varicocele include a volume difference between unaffected and affected testes, symptoms of pain or discomfort [33] as well as abnormalities in semen analysis [34]. However, obtaining semen from adolescents can be a sensitive topic and most often not available. Those with a persistent discrepancy between left and right testis size of more than 20 % [35, 36] over a period of 12 months and those with pain need varicocelectomy independent of patient age, Tanner stage or varicocele size [36]. The indications become confusing when discussing potential infertility as an indicator for repair. Inhibin B constitutes a reliable marker of Sertoli cell as well as spermatogenesis, in an attempt to identify early parameters of testicular dysfunction. Furthermore it could be a useful diagnostic marker of testicular damage caused by varicocele and could represent a new indication for varicocelectomy to avoid the future infertility problems in adulthood. Further studies are necessary to confirm the prognostic value of inhibin B levels in preserving the fertility and may give a guide to surgical intervention of these subset of patients.
References 1. Akbay E, Cayan S, Doruk E, Duce MN, Bozlu M (2000) The prevalence of varicocele and varicocele-related testicular atrophy in Turkish children and adolescents. BJU Int 86:490–493 2. Camoglio FS, Cervellione RM, Dipaola G, Balducci T, Giacomello L, Zanatta C, Forestieri C, Chironi C (2001) Idiopathic varicocele in children. Epidemiological study and surgical approach. Minerva Urol Nefrol 53:189–193 3. Al-Abbadi K, Smadi SA (2000) Genital abnormalities and groin hernias in elementary-school children in Aqaba:an epidemiological study. East Mediter Health J 6:293–298
Hernia 4. Hwang Y, Park SW (2009) Epidemiologic study of the prevalence and awareness of cryptorchidism, hydrocele, and varicocele in elementary schools in Gwangju. Korean J Urol 50:278–281 5. Belloli G, D’Agostino S, Pesce C, Fantuz E (1993) Varicocele in childhood and adolescence and other testicular anomalies: an epidemiological study. Pediatr Med Chir 15:159–162 6. Skoog SJ, Roberts KP, Goldstein M, Pryor JL (1997) The adolescent varicocele: what’s new with an old problem in young parients? Pediatrics 100:112–121 7. Male infertility best practice policy committee of the American Urological Association (2004) Report on varicocele and infertility. Fertil Steril 82(Suppl):S1 42–S145 8. Kass EJ, Freitas JE, Salisz JA, Steinert BW (1993) Pituitary gonadal dysfunction in adolescents with varicocele. Urology 42:179–181 9. Romeo C, Arrigo T, Impellizzeri P, Manganaro A, Antonuccio P, Di Pasquale G, Messina MF, Marseglia L, Formica I, Zuccarello B (2007) Altered serum inhibin B levels in adolescents with varicocele. J Ped Surg 42:390–394 10. Dubin L, Amelar RD (1970) Varicocele size and results of varicocele in selected subfertile men with varicocele. Fertil Steril 21:606–609 11. Marshall WA, Tanner JM (1970) Variations of pubertal changes in boys. Arch Dis Child 45:13–23 12. Trigo RV, Bergada I, Rey R, Ballerini MG, Bedecarras P, Bergada C, Gottlieb S, Campo S (2004) Altered serum profile of inhibin B, pro-alphaC and anti-mullerian hormone in prepubertal and pubertal boys with varicocele. Clin Endocrinol 60:758–764 13. Burger HG (1992) Inhibin. Reprod Med Rev 1:1–20 14. McLachan RI, Matsumoto AM, Burger HG, De Kretser DM, Bremner WJ (1988) Relative roles of follicle-stimulating hormone and luteinizing hormone in the control of inhibin secretion in normal men. J Clin Investig 82:880–884 15. Sharpe RM, Kerr JB, Maddocks S (1988) Evidence for a role of the Leydig cells in control of the intratesticular secretion of inhibin. Mol Cell Endocrinol 2–3:243–247 16. Andersson AM, Juul A, Petersen JH, Muller J, Groome NP, Skakkebaek NE (1997) Serum inhibin B in healthy pubertal and adolescent boys: relation to age, stage of puberty, and folliclestimulating hormone, luteinizing hormone, testosterone, and estradiol levels. J Clin Endocrinol Metab 82:3976–3981 17. Calsen E, Giwercman A, Skakkabaek NE, Keiding N (1992) Decreasing quality of semen. BMJ 305(6854):609–613 18. Illingworth PJ, Groome NP, Bryd W, Rainey WE, McNeilly AS, Mather JP, Bremner WJ (1996) Inhibin B: a likely candidate for the physiologically important form of inhibin in man. J Clin Endocrinol Metab 81:1321–1325 19. Ponchietti R, Raugai A, Grachi G (1987) Ultrastructural changes of Leydig cells in prepubertal varicocele. Acta Eur Fertil 18:347–348 20. Haans LC, Laven JS, Mali WP, Te Velde ER, Wensing CJ (1991) Testis volumes, semen quality, and hormonal patterns in adolescents with and without a varicocele. Fertil Steril 56:731–736
21. Steeno O, Knops J, Declerck L, Adimoelja A, Van de Voorde H (1976) Prevention of fertility disorders by detection and treatment of varicocele at school and college age. Andrologia 8:47–53 22. Lyon RP, Marshall S, Scott MP (1982) Varicocele in childhood and adolescence: implication in adult fertility. Urology 19:641–644 23. Paduch DA, Niedzielski J (1997) Repair versus observation in adolescent varicocele: a prospective study. J Urol 158:1128–1132 24. Sayfan J, Siplovich L, Koltun L, Benyamin N (1997) Varicocele treatment in pubertal boys prevents testicular growth arrest. J Urol 157:1456–1457 25. Guarino N, Tadini B, Bianchi M (2003) The adolescent varicocele: the crucial role of hormonal tests in selecting patients with testicular dysfunction. J Pediatr Surg 38:120–123 26. Liu MJ, Wang EH (2014) Impact of varicocele on semen quality and inhibin B concentration in serum and seminal plasma. Zhonghua Nan Ke Xue 20:44–47 27. Romeo C, Arrigo T, Impellizzeri P, Manganaro A, Antonuccio P, Di Pasquale G, Messina MF, Marseglia L, Formica I, Zuccarello B (2007) Altered serum inhibin B levels in adolescents with varicocele. J Ped Surg 42:390–394 28. Hayes FJ, Hall JE, Boepple PA, Crowley WF (1998) Clinical review 96: differential control of gonadotropin secretion in the human: endocrine role of inhibin. J Clin Endocrinol Metab 83:1835–1841 29. Foresta C, Bettella A, Rossato M, La Scala G, De Paoli M, Plebani M (1999) Inhibin B plasma concentration in oligozoospermic subjects before and after therapy with follicle stimulating hormone. Hum Reprod 14:906–912 30. Castro-Magana M, Angulo M, Canas A, Uy J (1990) Leydig cell function in adodescent boys with varicoceles. Arch Androl 24:73–79 31. Anyfantakis D, Symvoulakis EK, Barbounakis E, Kastanakis M, Athanasakis E, Blevrakis E, Kastanakis S (2014) A fatal case of seronegative, late-onset systemic lupus erythematosus presenting with motor sensory axonal polyneuropathy. Mod Rheumatol 24:858–861 32. Anyfantakis D, Symvoulakis EK (2011) Medical decision and patient’s preference: ‘much ethics’ and more trust always needed. Int J Med Sci 8:351–352 33. Schiff J, Kelly C, Goldstein M, Schlegel P, Poppas D (2005) Managing varicoceles in children: results with microsurgical varicocelectomy. BJU Int 95:399–402 34. Haddad NG, Houk CP, Lee PA (2014) Varicocele: a dilemma in adolescent males. Pediatr Endocrinol Rev 2:274–283 35. Diamond DA, Gargollo PC, Caldamone AA (2011) Current management principles for adolescent varicocele. Fert Steril 96:1294–1298 36. Waalkes R, Manea IF, Nijman JM (2012) Varicocele in adolescents: a review and guideline for the daily practice. Arch Esp Urol 65:859–871
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ORIGINAL ARTICLE
Impact of varicocele on biological markers of gonadal function E. Blevrakis • E. Chatzidarellis • D. Anyfantakis G. Sakellaris • M. Raissaki • O. Zoras • C. Mamoulakis • F. Sofras • E. Chrysos
•
Received: 14 August 2014 / Accepted: 20 February 2015 Ó Springer-Verlag France 2015
Abstract Purpose The aim of this study is to evaluate the potential effect of varicocele in the hormonal and clinical profile of adolescents. Methods Twenty adolescents at Tanner stage 4–5 with left varicocele were studied and compared with a control group of 20 healthy adolescents. All patients underwent ultrasonographic testicular volumetry as well as hormonal evaluation of inhibin B, testosterone, baseline and gonadotropin-releasing hormone stimulated, follicle-stimulating hormone as well as luteinizing hormone.
Statistical analysis was performed using the student’s t test with p value \0.05 taken as statistical significant. Results Patients with varicocele showed reduced levels of inhibin B compared to controls and a significant reduction in the testicular volume on the affected side. The response of luteinizing hormone to gonadotropin-releasing hormone stimulation was significantly higher in the varicocele group compared to the control group. Furthermore a significant inverse relationship of inhibin B compared to follicle-stimulating hormone was noted. Conclusion Serum inhibin B levels could represent a useful marker of Sertoli cell damage caused by varicocele. Keywords
E. Blevrakis (&) G. Sakellaris Department of Pediatric Surgery, University Hospital of Heraklion, Crete, Greece e-mail: [email protected] E. Chatzidarellis Department of Urology, Iaso General Hospital, Athens, Greece D. Anyfantakis Primary Health Care Centre of Kissamos, Chania, Crete, Greece M. Raissaki Department of Radiology, University Hospital of Heraklion, Crete, Greece O. Zoras Department of Surgical Oncology, University Hospital of Heraklion, Crete, Greece C. Mamoulakis F. Sofras Department of Urology, University Hospital of Heraklion, Crete, Greece E. Chrysos Department of General Surgery, University Hospital of Heraklion, Crete, Greece
Inhibin B Varicocele Gonadal function
Introduction Varicocele represents the abnormal dilatation of the pampiniform venous plexus inside the spermatic cord affecting about 15 % of the general male population and up to 40 % of males presenting to infertility clinics [1]. The condition is also detected in 2–11 % of prepubertal males [2–4] with a reported increase to 15–16 % in postpubertal adolescent males [5]. Although specific therapy is required in only 2 out of 10 affected males, the condition frequently leads to male infertility [6]. Pathophysiological mechanism through which varicocele results to testicular dysfunction has not yet been completely clarified. Potential etiollogies, include hormonal alterations, increase of non-collagenous proteins and immunoglobulins in the spermatic vein [6], production of toxic substances [7] as well as testicular hypoxia [6]. With regard to hormonal changes, it is still unknown whether these are the cause of gonadal damage.
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Hernia
Previous research efforts have attempted to identify parameters of testicular dysfunction, such as gonadotropin levels before and after stimulation with GnRH [8]. Inhibin B serum levels have been investigated in a group of young patients with varicocele at different Tanner developmental stages [9]. The authors concluded that the hormonal profile in prepubertal and pubertal males with varicocele may be altered. This may suggest an early involvement in seminiferous epithelial function [9]. The aim of the present study was to evaluate the hormonal profile of adolescents with varicocele in an attempt to use biochemical markers for selection of patients requiring surgical treatment and prevent ongoing testicular damage.
Materials and methods Over a 3 years period, 20 adolescent males with left unilateral varicocele were included in the study. All patients were referred for evaluation of an asymptomatic varicocele detected during routine pediatric examination. Diagnosis was confirmed through physical examination by a paediatric surgeon. Varicoceles were clinically classified according to the classification of Dubin and Amelar [10]. Pubertal stage was defined using Tanner’s criteria [11]. Testicular volume was determined by ultrasonography. Testicular growth arrest was considered when the testicular volume of the affected testicle was smaller than 2 ml compared to the contralateral testicle. The control group consisted of 20 age matched healthy adolescents of similar pubertal development stages and free of any endocrinological disorder. Endocrine function tests were performed in both varicocele adolescents and controls including FSH, LH, testosterone and inhibin B serum levels as well as stimulated LH and FSH, 30 and 60 min after intravenous administration of 100 lg GnRH. Inhibin B, FSH, LH and testosterone were measured by standardized radioimmunoassay techniques using ELISA commercial kits. Serum from each patient and control
Table 1 Mean and SD of inhibin B, FSH, LH and testis volumes from varicocele group and controls
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Results All males had a left-sided varicocele. The mean age of the patients was 13.8 years (range 12–17) and the mean age of the control group was 14.5 years (range 12–17). The biochemical markers of varicocele and control groups are summarized in Table 1. Patients with varicocele showed a significant reduction in the testicular volume of the affected side (12.9 ± 2.33 ml on the left vs 15.9 ± 2.32 ml on the right; p = 0.0001) (Fig. 1), whereas no significant difference was observed in the testis volume of the control group 17.1 ± 2.36 ml on the left vs 16.1 ± 2.39 ml on the right). No statistically significant differences in basal FSH, LH, testosterone and the maximal response of FSH were found between subjects with varicocele and the control group. The response of LH to GnRH stimulation was significantly higher in the varicocele group compared to the control group (20.8 ± 6.86 vs 15.8 ± 3.65 mIU/mL, respectively; p \ 0.05) (Fig. 2). Inhibin B levels were significantly lower in the varicocele group when compared to the age-matched controls (337 ± 33.6 vs 384 ± 35 pg/mL, respectively; p \ 0.05) (Fig. 3). Inhibin B levels in varicocele patients were positively correlated with testicular volume (r = 0.8646; p \ 0.05). No significant correlation was observed between inhibin B and testosterone or both baseline and stimulated LH. However, the correlation coefficient of inhibin B compared with FSH was negative and statistical significant (r = -0.826; p \ 0.05).
Varicocele group
Control group
p
Inhibin B (pg/mL)
337 ± 33.6
384 ± 35
0.0001
Testosterone (ng/dL) FSH baseline (mIU/mL)
335 ± 242 3.95 ± 1.39
340 ± 21.6 3.1 ± 1.28
0.49 (NS) 0.051 (NS)
FSH peak (mIU/mL)
7.73 ± 3.14
6.25 ± 2.16
0.09 (NS)
3.9 ± 1.65
3.47 ± 1.45
0.39 (NS)
LH peak (mIU/mL)
20.8 ± 6.86
15.8 ± 3.65
0.0066
Testicular volume R (mL)
15.9 ± 2.32
16.1 ± 2.39
0.083 (NS)
Testicular volume L (mL)
12.9 ± 2.33
17.1 ± 2.36
0.0001
LH baseline (mIU/mL)
NS indicates not significant
adolescents was frozen at -80 °C until assayed. Informed consent was obtained by both study groups and their guardians. Data are presented as the mean ± SD. Statistical analysis was performed using the Student’s t test for paired observations, with p value \0.05 taken as statistical significant and by calculation of the Pearson’s-Correlation coefficient.
LEFT TESTICULAR VOLUME (ml)
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LEFT TESTICULAR VOLUME
30
CONTROL GROUP
20
VARICOCELE GROUP
10 0
0
10
20
30
PATIENTS
Fig. 1 A plot of left testes volume in the varicocele patients and in control group. (12.9 ± 2.33 ml on the left testes in the varicocele patients vs 17.1 ± 2.36 ml on the left in the control group)
INHIBIN
Fig. 2 The response of LH to GnRH stimulation was significantly higher in the varicocele group than those of the control group (20.8 ± 6.86 mIU/mL in the varicocele group vs 15.8 ± 3.65 mIU/ mL in the control group)
500 450 400 350 300 250 200 150 100 50 0
INHIBIN B
CONTROL GROUP VARICOCELE GROUP
0
5
10
15
20
25
PATIENTS
Fig. 3 Inhibin B levels were significantly lower in the varicocele group when compared with the age-matched controls (337 ± 33.6 vs 384 ± 35 pg/mL)
Discussion Inhibins are glycoproteins predominantly produced in the gonads [12]. Inhibin B, composed of a and b subunits, is the physiologically relevant form of inhibin in males [13]. Both subunits are produced by Sertoli cells, regulated by FSH and unidentified factors by germ cells [14]. Leydig cell products may also influence inhibin synthesis in Sertoli cells [15]. The serum profile of inhibins has been determined in normal males from birth to puberty. After infancy, inhibin B serum concentration falls within the lower range of adult normal levels. At approximately the age of 10 years old inhibin B serum concentration starts to rise,
concomitantly with testicular volume and serum testosterone, reaching adult levels at Tanner stage 2 [16]. Issues of reproductive health of men in the general population such as the semen quality has received been subject of research efforts [17]. However, obtainment of semen samples in adults is difficult. Instead blood samples are easier to obtain than ejaculates. Therefore, serum biomarkers of spermatogenesis are of major interest for population studies. Circulating FSH has previously been used as a marker of spermatogenesis—has long been considered a valuable marker for Sertoli cell function, but FSH is influenced by hypothalamic function, as well as by testicular factors and steroid hormones. Serum Inhibin B was recently suggested as a promising, more direct serum marker of Sertoli cell function and spermatogenesis than FSH [18]. Early gonadal abnomarlities in patients with varicocele have been ascribed mainly to the interstitial compartment of the testis. Ponchietti et al. [19] described many of the ultrastructural changes in the testis of the peri-pubertal adolescent with a varicocele. These changes include a reduced number and atrophy of the Sertoli cells, germ cells and Leydig cells. They also reported fibrotic changes in the testicle, as well as sclerosis of the capillaries within the testis. A concomitant impairment of seminiferous tubule function has also been documented [20]; however, little information is available on the possible role of the Sertoli cell as a primary site of injury in these patients. Measurement of testicular volume is considered important in evaluating the adolescent with a varicocele. A significant size discrepancy between the testicles has become the main indication for surgical treatment. Steeno et al. [21] first observed a loss of testicular volume and/or a change in the consistency of the testis ipsilateral to the varicocele, most often the left testis, in about a third of children with Grade 2 varicoceles and in 80 % of boys with grade 3 varicoceles. Lyon et al. [22] reported a volume loss of the left testis in 77 % of adolescent boys with a clearly palpable left varicocele. In our study, we have found a significant reduction in the volume of the affected left testis in the varicocele group compared with both the contralateral testis and the testis control. Our findings are in accordance with those of preoperative testicular volumetric asymmetry by these investigators, and support the view that a large adolescent varicocele can be responsible for testicular growth retardadion. Trigo et al. [12] found a significantly lower volume in the affected left testis in patients with varicocele at Tanner stage 3–4, but not during the first and second stages of puberty. This could be explained as progressive damage caused by the varicocele upon seminiferous tubule function during the latter stages of puberty. The management of varicocele in adolescents is still controversial. One of the controversial issues is the
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existence of prognostic markers that would make it possible to decide for surgery and to determine, when necessary, the appropriate time to perform it [23, 24]. In our opinion, a better knowledge of the natural history of the disease might help to resolve this dilemma. For this reason, in our study we have evaluated the different biochemical parameters in pubertal patients, and by correlating them, we have made an attempt to analyze their potential usefulness. However, it is worth mentioning that since our patient population could not be followed up until adulthood, it was not possible to find clear prognostic markers of testicular damage. Another study found that the evaluation of testicular volume is not predictive for testicular dysfunction in adolescence [25]. Moreover, they could not find any statistical difference in testosterone serum levels between varicocele patients without semen abnormalities [25]. They demonstrated a significant increase in basal LH, FSH, and FSH after GnRH stimulation in varicocele patients with semen dysfunction [25]. According to the authors, only FSH and LH dosage could help in identifying those subjects at risk for future fertility problems [25]. In alignment with our findings a study among 95 infertile varicocele patients found lower concentration levels of inhibin B in the peripheral blood and seminal plasma compared to a control group of fertile males [26]. On the contrary, other studies have that in pubertal stages 4 and 5 inhibin B was lower in the varicocele group than in controls [12, 27]. We have also found reduced inhibin B serum levels in males with varicocele compared with controls. All varicocele patients were at Tanner stages 4–5. In these patients, we observed a significant positive correlation between inhibin B and testis volume. There was no significant correlation (either positive or negative) between inhibin B and LH and testosterone. However, the correlation coefficient of inhibin B compared with FSH achieve a statistical relevance. Inhibin B, as we mentioned, is a glycoprotein produced by Sertoli Cells and regulated by FSH and other factors produced by germ cells [28]. Inhibin B secretion depends on the interaction between Sertoli cells and germ cells. In particular, it is known that spermatids influence inhibin production and that these cells are sensitive to hyperthermia [29]. It is possible that the elevated scrotal temperature associated with varicocele has impaired spermatid function, thus reducing inhibin B production [30]. Alternatively reduced inhibin B serum levels in the varicocele group could result from early arrest of spermatogenesis or a depletion of germ cells. This might be expected to be associated with reduced testicular volume, which we have observed in the varicocele affected testis associated with reduced inhibin B serum levels. It has been shown that there is an increased nitrotyrosine concentration within the spermatic vein on the affected side
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in varicocele patients. This would be consistent with our findings. Further studies are necessary to support this hypothesis. In conclusion, although reduced serum inhibin B levels; a significant inverse relationship of inhibin B compared with FSH and higher response of LH to GnRH stimulation measured in adolescent boys with varicocele, we were not able to identify other abnormalities. Given the diagnostic uncertainty of medical science [31, 32] and on the basis of literature data and these findings, inhibin B might be useful diagnostic marker of testicular damage caused by varicocele and assist in the selection of patients for surgical treatment to prevent infertility problems. The deterioration of the testicular function in adolescents with varicocele is a progressive phenomenon that requires an intensive follow up to identify early signs of testicular dysfunction and to prevent further testicular damage. Surgical repair of varicocele is the most appropriate therapy to avoid infertility [33]. Absolute indications for varicocelectomy in adolescents with varicocele include a volume difference between unaffected and affected testes, symptoms of pain or discomfort [33] as well as abnormalities in semen analysis [34]. However, obtaining semen from adolescents can be a sensitive topic and most often not available. Those with a persistent discrepancy between left and right testis size of more than 20 % [35, 36] over a period of 12 months and those with pain need varicocelectomy independent of patient age, Tanner stage or varicocele size [36]. The indications become confusing when discussing potential infertility as an indicator for repair. Inhibin B constitutes a reliable marker of Sertoli cell as well as spermatogenesis, in an attempt to identify early parameters of testicular dysfunction. Furthermore it could be a useful diagnostic marker of testicular damage caused by varicocele and could represent a new indication for varicocelectomy to avoid the future infertility problems in adulthood. Further studies are necessary to confirm the prognostic value of inhibin B levels in preserving the fertility and may give a guide to surgical intervention of these subset of patients.
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