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Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia
FERTILITY AND STERILITY威 VOL. 76, NO. 6, DECEMBER 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia Santiago Brugo-Olmedo, M.D.,a Sabrina De Vincentiis, M.Sc.,a Juan C. Calamera, Ph.D.,b Fernando Urrutia, M.D.,a Florencia Nodar, M.Sc.,a and Anı´bal A. Acosta, M.D.a Center for Studies in Gynecology and Reproduction and Laboratory of Studies in Reproduction, Buenos Aires, Argentina
Objective: To establish the predictive value of serum inhibin B levels as an indicator of the presence of testicular spermatozoa in nonobstructive azoospermia, compared with the traditional serum FSH marker. Design: Prospective study. Setting: Private high-complexity reproductive center with university affiliation. Patient(s): Seventy-eight patients with nonobstructive azoospermia, 15 patients with obstructive azoospermia, and 10 fertile volunteers. Intervention(s): Blood samples, testicular sperm extraction, percutaneous epididymal sperm aspiration, and semen collection. Main Outcome Measure(s): Serum levels of inhibin B and FSH and presence of spermatozoa on TESE, PESA, or regular semen analysis. Result(s): Patients with nonobstructive azoospermia has significantly higher levels of serum FSH and significantly lower levels of inhibin B. Mean inhibin B serum levels were significantly higher in patients with nonobstructive azoospermia who had spermatozoa on TESE than in those in whom no spermatozoa were found (89.31 ⫾ 73.24 pg/mL vs. 19.23 ⫾ 22.34 pg/mL), but mean FSH serum levels did not have similar predictive power (21.37 ⫾ 12.92 IU/mL vs. 19.27 ⫾ 10.28 IU/mL). The cut-off level of inhibin B separating both groups, as determined by the receiver-operating characteristic curves, was ⬎53 pg/mL. Conclusion(s): Serum inhibin B level seems to be more accurate than serum FSH level in prediction of the presence of testicular spermatozoa in patients with nonobstructive azoospermia. (Fertil Steril威 2001;76: 1124 –9. ©2001 by American Society for Reproductive Medicine.) Key Words: Serum inhibin B, serum FSH, nonobstructive azoospermia, testicular spermatozoa Received February 21, 2001; revised and accepted June 11, 2001. Reprint requests: Santiago Brugo-Olmedo, M.D., Centro de Estudios en Ginecologı´a y Reproduccio´n, 1438 Viamonte (1055), Buenos Aires, Argentina (FAX: 54114371-7275; E-mail: [email protected]). a Center for Studies in Gynecology and Reproduction (CEGyR). b Laboratory of Studies in Reproduction (LER). 0015-0282/01/$20.00 PII S0015-0282(01)02866-7
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Different conventional markers of the presence of spermatozoa on testicular sperm extraction (TESE) in azoospermic men have been investigated. In obstructive azoospermia, testicular size, plasma FSH levels, and testicular histologic findings have proven to be useful, but no definitive markers have been found in men with nonobstructive azoospermia (1). A reliable method of predicting the presence or absence of clinically useful germinal epithelium (postmeiotic germ cells) would be highly desirable in patients with nonobstructive azoospermia, who currently must undergo TESE for confirmation. Inhibin is a glycoprotein hormone produced mainly by the gonads
under the influence of FSH and, in turn, is itself involved in the feedback regulation of the pituitary hormone (2). Circulating bioactive inhibin exists in at least two isoforms: A and B (an ␣ subunit linked by a disulphide bond to either a A or a B subunit, respectively) (3). In adult men, inhibin B is produced mainly in the Sertoli cells of testis under the influence of FSH (4), and circulating levels of inhibin A are undetectable. A negative correlation was recently observed between circulating levels of dimeric inhibin B and FSH in adult men (5). Early inhibin immunoassay results were unreliable owing to cross-reaction with monomeric pre-
cursor forms, lack of sensitivity, or inability to discriminate between A and B isoforms (6); however, these problems have been resolved (7). Inhibin B is a marker of testicular function. Serum levels of this hormone decrease when spermatogenic damage is caused by heat, cryptorchidism or pressure atrophy after prolonged efferent duct ligation. Other measures of Sertoli cell function, such as seminiferous tubule fluid production and androgen-binding protein secretion, also decrease in these situations (8). The value of peripheral inhibin B levels in predicting the presence of germinal epithelium, including spermatozoa, in the testes of patients with nonobstructive azoospermia is not clearly established. In a small study, Ballesca´ et al. (9) found that serum inhibin B level was a good predictor of the presence of testicular spermatozoa; in contrast, von Eckardstein et al. (10) concluded that this variable cannot accurately predict TESE outcome. We sought to determine whether basal serum levels of inhibin B can be used to predict the presence of testicular spermatozoa in azoospermic patients and whether these levels are more reliable than serum FSH measurement.
MATERIALS AND METHODS Patients Between August 1997 and August 2000, 72 patients classified on clinical grounds as having nonobstructive azoospermia and no identifiable etiologic factors (idiopathic) and 6 patients with identifiable possible causes (systemic chemotherapy for Hodgkin disease in 1 patient and the Klinefelter syndrome with a XXY sex chromosome aneuploidy in 5 patients) were studied. During the same period, 15 patients with obstructive azoospermia and 10 fertile volunteers were enrolled as positive controls. Exclusion criteria for patients and controls were history of chronic diseases, alcoholism, drug addiction, use of anabolic steroids, or use of hormonal treatment.
Percutaneous Epididymal Sperm Aspiration Percutaneous epididymal sperm aspiration (PESA) was done according to the method of Craft et al. (11), with some modifications. Briefly, PESA was performed under light anesthesia; for aspiration, at least four 1-mL syringes were used per epididymis. The syringes contained 0.3 mL of HEPES buffer– human tubal fluid (H-HTF) medium (Irvine Scientific Laboratories, Santa Ana, CA) supplemented with 1% bovine serum albumin (BSA) (A-4503; Sigma Laboratories, St. Louis, MO). Each syringe was connected to a 21-gauge needle and was directed to the head or the corpus of the epididymis after the testes were immobilized; by holding the testes stable beneath the thumb and index finger, which were placed above them so that the surgeon was able to feel the epididymis. Suction was applied several times FERTILITY & STERILITY威
with each syringe and was stopped regardless of whether fluid was observed to come out of the epididymis. All syringes were sent immediately to the laboratory. The suspension was deposited in a small Petri dish (FALCON [catalog no. 3652;] Becton-Dickinson, Lincoln Park, NJ), 1 mL of H-HTF with 1% BSA was added, and a droplet was examined under an inverted microscope (Nikon Diaphot; Nikon Corp., Tokyo, Japan) with a heated platform (Nikon Corp.) to search for spermatozoa. The suspension was then pulled into a 15-mL tube (FALCON [catalog no. 2196]; Becton-Dickinson) and centrifuged for 5 minutes at 1,600 rpm. The pellet was resuspended in 100 –500 L of H-HTF and 1% BSA and incubated (5% CO2, 5% O2, 90% N2, humidified atmosphere at 37°C) until use.
Testicular Sperm Extraction Multiple testicular biopsies were performed under local anesthesia. Tissue samples were placed in tubes (FALCON [catalog no. 2001]; Becton-Dickinson) containing 2 mL of H-HTF supplemented with 1% BSA and were sent to the laboratory. The tissue that was obtained was dispersed with the help of two sterile slides in a small Petri dish. Testicular tissue was examined under an inverted microscope with a heated platform to search for spermatozoa. One mL of HHTF with 1% BSA was then added, and the tissue was carefully dispersed. The suspension was centrifuged for 5 minutes at 1,600 rpm. The pellet was resuspended in 100 – 500 L of H-HTF with 1% BSA. The suspension was again checked under the microscope and incubated (5% CO2, 5% O2, 90% N2, humidified atmosphere at 37°C) until use.
Cryopreservation of Epididymal and Testicular Spermatozoa We used the cryopreservation technique of Romero et al. (12), with slight modification. Briefly, sperm suspension was diluted in a 1:1 ratio in test yolk buffer cryopreservation medium containing glycerol (catalog no. 9971; Irvine Scientific). The cryoprotectant was added very slowly. After gently homogenization, a sterile glass Pasteur pipette was used to deposit the mixture in approximately 100 L droplets on a dry ice surface. Freezing of droplets (“pills”) occurred in less than 1 minute. The frozen pills were placed into precooled 1.2-mL cryovials (COSTAR [catalog no. 2012]; Biofreeze Vial, Corning Costar Corp., Cambridge, MA), plunged into liquid nitrogen (⫺196°C), and stored.
Hormone Assays Blood samples were drawn from an antecubital vein between 8:00 and 10:00 A.M.; in patients with nonobstructive or obstructive azoospermia, samples were obtained before TESE or PESA was done because tissue damage may modify inhibin B levels. The samples were centrifuged at 2,000 rpm for 5–10 minutes, and the serum was separated, extracted, and stored at ⫺20°C until processing. A chemoluminescence method (Access Immunoassay System; Beckman-Coulter, Chaska, MN) was used to mea1125
sure. The Access immunoassay is a sequential, two-step immunoenzymatic (sandwich) technique. The sample is added to a reaction vessel with paramagnetic particles coated with goat antimouse:mouse anti-h-FSH complexes and Trisbuffered saline with protein. The sensitivity of the method is ⬍0.2 mIU/mL. The intraassay coefficient of variation was found to be 3.1%; nevertheless, the intraassay coefficient of variation depends on low, medium, or high circulating hormone levels and was found to be 3.5%, 3.1%, and 4.3%, respectively. Dimeric inhibin B was analyzed by using a solid-phase sandwich enzyme-linked immunosorbent assay (Serotec; Serotec Ltd., Oxford, United Kingdom) according to the manufacturer’s instructions. The sensitivity of the method is ⬍15 pg/mL. The kit detects dimeric inhibin B and demonstrates minimal cross-reactivity with the proalpha C subunit or with activins. It has low cross-reactivity with inhibin A (approximately 1%). Reproducibility interplate and intraplate coefficient of variations are ⬍7%.
Consent All couples signed a written consent form for each procedure performed (TESE, PESA, sperm cryopreservation, and ICSI). Institutional review board approval was not requested because this work is a prospective case study.
Genetic Screening All couples undergoing ICSI were asked to undergo standard peripheral blood karyotyping before the procedure.
Statistical Analysis Raw data were reviewed manually for values that could help identify and predict presence or absence of testicular sperm on TESE (Tables 1 and 2). Data were analyzed by using one-way analysis of variance, the nonparametric Kruskal–Wallis test (StatPlus software, version b384; StatSoft, Inc., TX), and receiver-operating characteristic (ROC) curves (Graph ROC for Windows, version 2.0; KairistoPoola). The ROC analysis is a method of establishing the best cut-off value of a variable to discriminate between two outcomes, such as normal vs. abnormal test results. Once the best delimiting value was established, optimum sensitivity, specificity, positive predictive value, and negative predictive value were calculated.
RESULTS Testicular sperm extraction was successful in 30 of 72 (41.7%) men with idiopathic nonobstructive azoospermia and 3 of 6 (50%) men with identifiable causes of nonobstructive azoospermia. All men with obstructive azoospermia had spermatozoa on PESA or TESE. Tables 1 and 2 show individual raw data on inhibin B serum levels. Mean serum FSH levels were significantly lower in men with idiopathic nonobstructive azoospermia than in men with identifiable causes of nonobstructive azoospermia (P⬍.05). 1126 Brugo-Olmedo et al.
TABLE 1 Data on individual men with idiopathic nonobstructive azoospermia in whom testicular sperm extraction failed. Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
FSH level (IU/mL)
Inhibin B level (pg/mL)
16.3 16.8 18.9 15.9 4.9 24.86 19.0 14.0 30.5 11.2 9.9 34.0 30.4 24.8 20.9 28.21 31.2 20.3 31.4 10.5 18.0 16.3 15.6 47.8 35.4 14.1 8.0 17.0 36.3 23.7 7.49 9.6 8.6 24.1 10.5 10.7 7.8 10.9 40.0 10.8 7.53 15.3
ND ND ND ND ND ND ND ND ND ND ND ND 0.23 2.0 4.6 5.2 8.0 8.0 8.0 10.0 10.9 13.0 13.4 13.48 14.91 19.0 22.72 24.0 24.0 28.0 31.0 31.0 35.0 35.0 36.0 36.0 40.0 52.3 53.0 61.0 80.0 88.0
Note: ND ⫽ not detectable. Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
Mean serum levels of inhibin B did not differ between the groups, perhaps because of the small number of patients in the latter group. Serum levels of FSH and inhibin B were also compared in the other two study groups; results are shown in Table 3. Patients with idiopathic nonobstructive azoospermia had similar serum FSH levels regardless of whether spermatozoa were present on TESE (21.37 ⫾ 12.92 IU/mL in those with spermatozoa on TESE vs. 19.27 ⫾ 10.28 IU/mL in those without spermatozoa), but those with spermatozoa had significantly higher levels of serum inhibin
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TABLE 2 Data on individual men with idiopathic nonobstructive azoospermia in whom testicular sperm extraction was successful. Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
FSH level (IU/mL)
Inhibin B level (pg/mL)
29.75 32.2 46.3 28.2 32.3 31.9 20.6 19 33.9 12.1 1.9 13 35 15.5 21.5 16.45 20 36.7 52 16.76 3.5 20.7 9.76 2.8 30 23.3 9 6.4 18 2.7
ND ND 4.0 17.04 20.0 23.0 27.0 36.0 43.0 51.0 60.15 62.0 62.0 66.5 77.0 80.0 80.0 84.0 86.0 86.6 100.0 115.0 125.0 139.89 141.0 160.0 176.0 222.0 226.0 309.0
Note: ND ⫽ not detectable. Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
B than did those without spermatozoa (89.31 ⫾ 73.24 pg/mL vs. 19.23 ⫾ 22.34 pg/mL; P⫽.00009) (Tables 4 and 5).
To analyze further the diagnostic accuracy (predictive value of sperm retrieval) of inhibin B to discriminate between success and failure with TESE, the area under the ROC curves were determined for serum FSH and inhibin B (Fig. 1). The area under the ROC for inhibin B in predicting the likelihood of successful TESE was significantly higher than that for FSH. The ROC curve analysis was also used to determine the best threshold value for inhibin B in predicting success or failure of TESE. The inhibin B value that discriminated best between success and failure of TESE was 53 pg/mL (sensitivity, 91.11% [CI, 0.786 – 0.9764]; specificity, 75.86% [CI, 0.6276 – 0.8618]). Positive and negative predictive values were 0.7455 and 0.9167, respectively.
DISCUSSION Recent reports have suggested that recovery of testicular spermatozoa may be possible in more than 50% of men with nonobstructive azoospermia, regardless of clinical variables, size of the testes, or plasma FSH concentration (13–15). Fresh and cryopreserved testicular spermatozoa from azoospermic men have been used successfully in ICSI (16), which allows TESE to be performed before ovarian stimulation. Discovery of a reliable marker that predicts and correlates well with the presence of testicular spermatozoa on TESE in patients with nonobstructive azoospermia remains a high diagnostic priority. Follicle-stimulating hormone is currently regarded as the most important endocrine variable in evaluation of male testicular function (17). Its secretion is suppressed by the testicular hormone inhibin, which is produced in Sertoli cells and is therefore a serum marker for Sertoli cell function. Castration results in undetectable inhibin B levels, indicating that circulating inhibin B is produced by the testes (4). Nevertheless, deKretser et al. (18) postulated that inhibin levels did not differ in fertile controls and subfertile men with testicular disorders; this finding may have resulted from
TABLE 3 Serum FSH and inhibin B values. Group Idiopathic nonobstructive azoospermia (I) Nonobstructive azoospermia of identifiable (II) Obstructive azoospermia (III) Fertile volunteers (IV)
No. of patients
Serum FSH level (IU/mL)
Serum inhibin B level (pg/mL)
72 6 15 10
20.15 ⫾ 11.42a 36.13 ⫾ 9.66b 3.95 ⫾ 1.63c 7.37 ⫾ 8.18
48.43 ⫾ 60.74d 13.33 ⫾ 24.91e 156.83 ⫾ 80.14f 231.20 ⫾ 114.62
Note: Values are means ⫾ SD. a Group I vs. group II, P⫽.041; group I vs. group III, P⬍.0005; group I vs. group IV, P⫽.004. b Group II vs. group III, P⫽.002; group II vs. group IV, P⫽.001. c Group III vs. group IV, P⫽0.725. d Group I vs. group II, P⫽.086; group I vs. group III, P⫽.001; group I vs. group IV, P⫽.003. e Group II vs. group III, P⬍.0005; group II vs. group IV, P⫽.001. f Group III vs. group IV, P⫽.416. Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
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TABLE 4 Serum FSH and inhibin B values in men with idiopathic nonobstructive azoospermia. Group Idiopathic nonobstructive azoospermia with successful TESE with failed TESE
No. of patients (%)
Serum FSH level (IU/mL)
Serum inhibin B level (pg/mL)
72 (100) 30 (41.7) 42 (58.3)
20.15 ⫾ 11.42 21.37 ⫾ 12.92a 19.27 ⫾ 10.28a
48.43 ⫾ 60.74 89.31 ⫾ 73.24b 19.23 ⫾ 22.34b
Note: Values are means ⫾ SD. TESE ⫽ testicular sperm extraction. a Successful TESE vs. failed TESE, P⫽.97. b Successful TESE vs. failed TESE, P⫽0.00009. Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
the low specificity of the inhibin assay used. The few reports on serum levels of inhibin B relative to quality of spermatogenesis (4, 5, 19 –21) seem to strongly indicate that inhibin B is an important marker of the competence of Sertoli cells and of the presence of spermatogenesis in the human. Pierik et al. (21) found a significant correlation between inhibin B levels and sperm concentration, sperm count, and testicular volume. They provided strong evidence that inhibin B is a valuable marker of the presence of spermatogenesis by showing a significant positive correlation between inhibin B levels and the most accurate assessment of spermatogenesis in their setting, the testicular biopsy score. Few reports indicated that serum inhibin B level is a predictor of the presence of testicular spermatozoa in azoospermic men, and results have been conflicting (9, 10). We compared serum FSH and inhibin B level in positive controls (fertile males with normozoospermia and patients with obstructive azoospermia) and men with idiopathic nonobstructive azoospermia. We also studied these variables in subgroups of nonobstructive azoospermic men with or without spermatozoa on TESE.
Patients with spermatozoa on biopsy have undeniable evidence of focal spermatogenesis; however, men with negative findings may still have areas of focal spermatogenesis that could have been missed on biopsy. Therefore, use of TESE results as the end point and the gold standard to be correlated with serum FSH or inhibin B levels as predictive markers is somewhat unreliable. Prediction of the presence or absence of germinal epithelium in abnormal testes from azoospermic patients is of utmost importance in planning treatment. If the couple decides to proceed with the attempt despite the available in-
FIGURE 1 Receiver-operating characteristic curves of inhibin B and FSH in discriminating success or failure of testicular sperm extraction in men with idiopathic nonobstructive azoospermia.
TABLE 5 Percentage of men with idiopathic nonobstructive azoospermia according to FSH and inhibin B cut-off levels. FSH serum level Good prognosis: FSH ⬍ 19 No. of patients: 38 13 (34.2%); successful TESE 25 (65.8%); failed TESE
Bad prognosis: FSH ⱖ 19 No. of patients: 34 17 (50%); successful TESE 17 (50%); failed TESE
Inhibin B serum level Good prognosis: IB ⬎ 53 No. of patients: 24 20 (83.3%); successful TESE 4 (16.7%); failed TESE
Bad prognosis: IB ⬍ 53 No. of patients: 48 10 (20.8%); successful TESE 38 (79.2%); failed TESE
Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
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Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
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formation, biopsy can be performed before the stimulation cycle in the female partner is started; thereby sparing her a possibly unnecessary procedure. If sperm is found unexpectedly in the testicular tissue, it can be frozen for ICSI in the future, avoiding a second biopsy in the male partner. The accuracy of the basal serum inhibin B level in predicting presence or absence of germinal epithelium in patients with nonobstructive azoospermia must be evaluated further in a larger sample. Individual serum levels of inhibin B serum cannot currently be used as the definitive test for evaluating the presence or absence of spermatozoa in testicles of men with nonobstructive azoospermia; in these patients, TESE remains the most accurate diagnostic method.
8. 9.
10.
11.
12. 13. 14.
Acknowledgments: The authors thank L. Vargas, M.D., M. Albamonte, M.D., and V. Bertollino, M.D. for help with data collection and Ms. N. Durand, our librarian, for professional help.
15.
16.
References 1. Mulhall JP, Burgess CM, Cunningham D, Carson R, Harris D, Oates RD. Presence of mature sperm in testicular parenchyma of men with nonobstructive azoospermia: prevalence and predictive factors. Urology 1997;49:91– 6. 2. Burger HG, Igarashi M. Inhibin: definition and nomenclature, including related substances. J Clin Endocrinol Metab 1988;66:885– 6. 3. Robertson DM, Sullivan J, Watson M, Cahir N. Inhibin forms in human plasma. J Endocrinol 1995;144:261–9. 4. Anawalt BD, Bebb RA, Matsumoto AM, Groome NP, Illingworth PJ, McNeilly AS, et al. Serum Inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endocrinol Metab 1996;81:3341– 45. 5. Illingworth PJ, Groome NP, Byrd W, Rainey WE, McNeilly AS, Mather JP, et al. Inhibin-B: a likely candidate for the physiologically important form of inhibin in men. J Clin Endocrinol Metab 1996;81: 1321–5. 6. Robertson DM, Giacometti M, Foulds LM, Lahnstein J, Goss NH, Hearn MT, et al. Isolation of inhibin alpha-subunit precursor proteins from bovine follicular fluid. Endocrinology 1989;125:2141–9. 7. Groome NO, Illingworth PJ, O’Brien M, Pai R, Rodger FE, Mather JP,
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et al. Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 1996;81:1401–5. Au CL, Robertson DM, DeKretser DM. In vitro bioassay of inhibin in testes of normal and cryptorchid rats. Endocrinology 1983;112:239 – 44. Ballesca´ J, Balasch J, Calafell JM, Alvarez R, Fa´ bregues F, Martinez de Osaba MJ, et al. Serum Inhibin B determination is predictive of successful testicular sperm extraction in men with nonobstructive azoospermia. Hum Reprod 2000;15:1734 – 8. von Eckardstein S, Simoni M, Bergmann M, Weinbauer GF, Gassner P, Schepers AG, et al. Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. J Clin Endocrinol Metab 1999;84:2496 –501. Craft I, Tsirigotis M, Bennett V, Taranissi M, Khalifa Y, Hogewind G, et al. Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertil Steril 1995;63:1038 – 42. Romero J, Remohı´ J, Minguez Y, Rubio C, Pellicer A, Gil-Salom M. Fertilization after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Fertil Steril 1996;65:877–9. Cha KY, Oum KB, Kim HJ. Approaches for obtaining sperm in patients with male factor infertility. Fertil Steril 1997;67:985–95. Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber S, et al. Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum Reprod 1997;12:80 – 6. Ezeh IUO, Moore HDM, Cooke ID. Correlation of testicular sperm extraction with morphological, biophysical and endocrine profiles in men with azoospermia due to primary gonadal failure. Hum Reprod 1998;13:3066 –74. Palermo GD, Schlegel PN, Hariprashad JJ, Ergun B, Mielnik A, Zaninovic N, et al. Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum Reprod 1999;14: 741– 8. Nieschlag E. Care for the infertile male. Clin Endocrinol (Oxf) 1993; 38:123–33. deKretser DM, McLachlan RI, Robertson DM, Burger HG. Serum inhibin levels in normal men and men with testicular disorders. J Endocrinol 1989;120:517–23. Jensen TK, Andersson AM, Hjollund NHI, Scheike T, Kolstad H, Giwercman A, et al. Inhibin B as a serum marker of spermatogenesis: correlation to differences in sperm concentration and follicle-stimulating hormone levels. A study of 349 Danish men. J Clin Endocrinol Metab 1997;82:4059 – 63. Andersson AM, Juul A, Petersen JH, Muller J, Groome NP, Skakkebaek NE. Serum inhibin B in healthy pubertal and adolescent boys: relation to the age stage of puberty, and follicle-stimulating hormone, testosterone, and estradiol levels. J Clin Endocrinol Metab 1997;82: 3976 – 81. Pierik FH, Vreeburg JTM, Stijnen T, De Jong FA, Waber RFA. Serum inhibin B as a marker of spermatogenesis. J Clin Endocrinol Metab 1998;83:3110 – 4.
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Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia Santiago Brugo-Olmedo, M.D.,a Sabrina De Vincentiis, M.Sc.,a Juan C. Calamera, Ph.D.,b Fernando Urrutia, M.D.,a Florencia Nodar, M.Sc.,a and Anı´bal A. Acosta, M.D.a Center for Studies in Gynecology and Reproduction and Laboratory of Studies in Reproduction, Buenos Aires, Argentina
Objective: To establish the predictive value of serum inhibin B levels as an indicator of the presence of testicular spermatozoa in nonobstructive azoospermia, compared with the traditional serum FSH marker. Design: Prospective study. Setting: Private high-complexity reproductive center with university affiliation. Patient(s): Seventy-eight patients with nonobstructive azoospermia, 15 patients with obstructive azoospermia, and 10 fertile volunteers. Intervention(s): Blood samples, testicular sperm extraction, percutaneous epididymal sperm aspiration, and semen collection. Main Outcome Measure(s): Serum levels of inhibin B and FSH and presence of spermatozoa on TESE, PESA, or regular semen analysis. Result(s): Patients with nonobstructive azoospermia has significantly higher levels of serum FSH and significantly lower levels of inhibin B. Mean inhibin B serum levels were significantly higher in patients with nonobstructive azoospermia who had spermatozoa on TESE than in those in whom no spermatozoa were found (89.31 ⫾ 73.24 pg/mL vs. 19.23 ⫾ 22.34 pg/mL), but mean FSH serum levels did not have similar predictive power (21.37 ⫾ 12.92 IU/mL vs. 19.27 ⫾ 10.28 IU/mL). The cut-off level of inhibin B separating both groups, as determined by the receiver-operating characteristic curves, was ⬎53 pg/mL. Conclusion(s): Serum inhibin B level seems to be more accurate than serum FSH level in prediction of the presence of testicular spermatozoa in patients with nonobstructive azoospermia. (Fertil Steril威 2001;76: 1124 –9. ©2001 by American Society for Reproductive Medicine.) Key Words: Serum inhibin B, serum FSH, nonobstructive azoospermia, testicular spermatozoa Received February 21, 2001; revised and accepted June 11, 2001. Reprint requests: Santiago Brugo-Olmedo, M.D., Centro de Estudios en Ginecologı´a y Reproduccio´n, 1438 Viamonte (1055), Buenos Aires, Argentina (FAX: 54114371-7275; E-mail: [email protected]). a Center for Studies in Gynecology and Reproduction (CEGyR). b Laboratory of Studies in Reproduction (LER). 0015-0282/01/$20.00 PII S0015-0282(01)02866-7
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Different conventional markers of the presence of spermatozoa on testicular sperm extraction (TESE) in azoospermic men have been investigated. In obstructive azoospermia, testicular size, plasma FSH levels, and testicular histologic findings have proven to be useful, but no definitive markers have been found in men with nonobstructive azoospermia (1). A reliable method of predicting the presence or absence of clinically useful germinal epithelium (postmeiotic germ cells) would be highly desirable in patients with nonobstructive azoospermia, who currently must undergo TESE for confirmation. Inhibin is a glycoprotein hormone produced mainly by the gonads
under the influence of FSH and, in turn, is itself involved in the feedback regulation of the pituitary hormone (2). Circulating bioactive inhibin exists in at least two isoforms: A and B (an ␣ subunit linked by a disulphide bond to either a A or a B subunit, respectively) (3). In adult men, inhibin B is produced mainly in the Sertoli cells of testis under the influence of FSH (4), and circulating levels of inhibin A are undetectable. A negative correlation was recently observed between circulating levels of dimeric inhibin B and FSH in adult men (5). Early inhibin immunoassay results were unreliable owing to cross-reaction with monomeric pre-
cursor forms, lack of sensitivity, or inability to discriminate between A and B isoforms (6); however, these problems have been resolved (7). Inhibin B is a marker of testicular function. Serum levels of this hormone decrease when spermatogenic damage is caused by heat, cryptorchidism or pressure atrophy after prolonged efferent duct ligation. Other measures of Sertoli cell function, such as seminiferous tubule fluid production and androgen-binding protein secretion, also decrease in these situations (8). The value of peripheral inhibin B levels in predicting the presence of germinal epithelium, including spermatozoa, in the testes of patients with nonobstructive azoospermia is not clearly established. In a small study, Ballesca´ et al. (9) found that serum inhibin B level was a good predictor of the presence of testicular spermatozoa; in contrast, von Eckardstein et al. (10) concluded that this variable cannot accurately predict TESE outcome. We sought to determine whether basal serum levels of inhibin B can be used to predict the presence of testicular spermatozoa in azoospermic patients and whether these levels are more reliable than serum FSH measurement.
MATERIALS AND METHODS Patients Between August 1997 and August 2000, 72 patients classified on clinical grounds as having nonobstructive azoospermia and no identifiable etiologic factors (idiopathic) and 6 patients with identifiable possible causes (systemic chemotherapy for Hodgkin disease in 1 patient and the Klinefelter syndrome with a XXY sex chromosome aneuploidy in 5 patients) were studied. During the same period, 15 patients with obstructive azoospermia and 10 fertile volunteers were enrolled as positive controls. Exclusion criteria for patients and controls were history of chronic diseases, alcoholism, drug addiction, use of anabolic steroids, or use of hormonal treatment.
Percutaneous Epididymal Sperm Aspiration Percutaneous epididymal sperm aspiration (PESA) was done according to the method of Craft et al. (11), with some modifications. Briefly, PESA was performed under light anesthesia; for aspiration, at least four 1-mL syringes were used per epididymis. The syringes contained 0.3 mL of HEPES buffer– human tubal fluid (H-HTF) medium (Irvine Scientific Laboratories, Santa Ana, CA) supplemented with 1% bovine serum albumin (BSA) (A-4503; Sigma Laboratories, St. Louis, MO). Each syringe was connected to a 21-gauge needle and was directed to the head or the corpus of the epididymis after the testes were immobilized; by holding the testes stable beneath the thumb and index finger, which were placed above them so that the surgeon was able to feel the epididymis. Suction was applied several times FERTILITY & STERILITY威
with each syringe and was stopped regardless of whether fluid was observed to come out of the epididymis. All syringes were sent immediately to the laboratory. The suspension was deposited in a small Petri dish (FALCON [catalog no. 3652;] Becton-Dickinson, Lincoln Park, NJ), 1 mL of H-HTF with 1% BSA was added, and a droplet was examined under an inverted microscope (Nikon Diaphot; Nikon Corp., Tokyo, Japan) with a heated platform (Nikon Corp.) to search for spermatozoa. The suspension was then pulled into a 15-mL tube (FALCON [catalog no. 2196]; Becton-Dickinson) and centrifuged for 5 minutes at 1,600 rpm. The pellet was resuspended in 100 –500 L of H-HTF and 1% BSA and incubated (5% CO2, 5% O2, 90% N2, humidified atmosphere at 37°C) until use.
Testicular Sperm Extraction Multiple testicular biopsies were performed under local anesthesia. Tissue samples were placed in tubes (FALCON [catalog no. 2001]; Becton-Dickinson) containing 2 mL of H-HTF supplemented with 1% BSA and were sent to the laboratory. The tissue that was obtained was dispersed with the help of two sterile slides in a small Petri dish. Testicular tissue was examined under an inverted microscope with a heated platform to search for spermatozoa. One mL of HHTF with 1% BSA was then added, and the tissue was carefully dispersed. The suspension was centrifuged for 5 minutes at 1,600 rpm. The pellet was resuspended in 100 – 500 L of H-HTF with 1% BSA. The suspension was again checked under the microscope and incubated (5% CO2, 5% O2, 90% N2, humidified atmosphere at 37°C) until use.
Cryopreservation of Epididymal and Testicular Spermatozoa We used the cryopreservation technique of Romero et al. (12), with slight modification. Briefly, sperm suspension was diluted in a 1:1 ratio in test yolk buffer cryopreservation medium containing glycerol (catalog no. 9971; Irvine Scientific). The cryoprotectant was added very slowly. After gently homogenization, a sterile glass Pasteur pipette was used to deposit the mixture in approximately 100 L droplets on a dry ice surface. Freezing of droplets (“pills”) occurred in less than 1 minute. The frozen pills were placed into precooled 1.2-mL cryovials (COSTAR [catalog no. 2012]; Biofreeze Vial, Corning Costar Corp., Cambridge, MA), plunged into liquid nitrogen (⫺196°C), and stored.
Hormone Assays Blood samples were drawn from an antecubital vein between 8:00 and 10:00 A.M.; in patients with nonobstructive or obstructive azoospermia, samples were obtained before TESE or PESA was done because tissue damage may modify inhibin B levels. The samples were centrifuged at 2,000 rpm for 5–10 minutes, and the serum was separated, extracted, and stored at ⫺20°C until processing. A chemoluminescence method (Access Immunoassay System; Beckman-Coulter, Chaska, MN) was used to mea1125
sure. The Access immunoassay is a sequential, two-step immunoenzymatic (sandwich) technique. The sample is added to a reaction vessel with paramagnetic particles coated with goat antimouse:mouse anti-h-FSH complexes and Trisbuffered saline with protein. The sensitivity of the method is ⬍0.2 mIU/mL. The intraassay coefficient of variation was found to be 3.1%; nevertheless, the intraassay coefficient of variation depends on low, medium, or high circulating hormone levels and was found to be 3.5%, 3.1%, and 4.3%, respectively. Dimeric inhibin B was analyzed by using a solid-phase sandwich enzyme-linked immunosorbent assay (Serotec; Serotec Ltd., Oxford, United Kingdom) according to the manufacturer’s instructions. The sensitivity of the method is ⬍15 pg/mL. The kit detects dimeric inhibin B and demonstrates minimal cross-reactivity with the proalpha C subunit or with activins. It has low cross-reactivity with inhibin A (approximately 1%). Reproducibility interplate and intraplate coefficient of variations are ⬍7%.
Consent All couples signed a written consent form for each procedure performed (TESE, PESA, sperm cryopreservation, and ICSI). Institutional review board approval was not requested because this work is a prospective case study.
Genetic Screening All couples undergoing ICSI were asked to undergo standard peripheral blood karyotyping before the procedure.
Statistical Analysis Raw data were reviewed manually for values that could help identify and predict presence or absence of testicular sperm on TESE (Tables 1 and 2). Data were analyzed by using one-way analysis of variance, the nonparametric Kruskal–Wallis test (StatPlus software, version b384; StatSoft, Inc., TX), and receiver-operating characteristic (ROC) curves (Graph ROC for Windows, version 2.0; KairistoPoola). The ROC analysis is a method of establishing the best cut-off value of a variable to discriminate between two outcomes, such as normal vs. abnormal test results. Once the best delimiting value was established, optimum sensitivity, specificity, positive predictive value, and negative predictive value were calculated.
RESULTS Testicular sperm extraction was successful in 30 of 72 (41.7%) men with idiopathic nonobstructive azoospermia and 3 of 6 (50%) men with identifiable causes of nonobstructive azoospermia. All men with obstructive azoospermia had spermatozoa on PESA or TESE. Tables 1 and 2 show individual raw data on inhibin B serum levels. Mean serum FSH levels were significantly lower in men with idiopathic nonobstructive azoospermia than in men with identifiable causes of nonobstructive azoospermia (P⬍.05). 1126 Brugo-Olmedo et al.
TABLE 1 Data on individual men with idiopathic nonobstructive azoospermia in whom testicular sperm extraction failed. Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
FSH level (IU/mL)
Inhibin B level (pg/mL)
16.3 16.8 18.9 15.9 4.9 24.86 19.0 14.0 30.5 11.2 9.9 34.0 30.4 24.8 20.9 28.21 31.2 20.3 31.4 10.5 18.0 16.3 15.6 47.8 35.4 14.1 8.0 17.0 36.3 23.7 7.49 9.6 8.6 24.1 10.5 10.7 7.8 10.9 40.0 10.8 7.53 15.3
ND ND ND ND ND ND ND ND ND ND ND ND 0.23 2.0 4.6 5.2 8.0 8.0 8.0 10.0 10.9 13.0 13.4 13.48 14.91 19.0 22.72 24.0 24.0 28.0 31.0 31.0 35.0 35.0 36.0 36.0 40.0 52.3 53.0 61.0 80.0 88.0
Note: ND ⫽ not detectable. Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
Mean serum levels of inhibin B did not differ between the groups, perhaps because of the small number of patients in the latter group. Serum levels of FSH and inhibin B were also compared in the other two study groups; results are shown in Table 3. Patients with idiopathic nonobstructive azoospermia had similar serum FSH levels regardless of whether spermatozoa were present on TESE (21.37 ⫾ 12.92 IU/mL in those with spermatozoa on TESE vs. 19.27 ⫾ 10.28 IU/mL in those without spermatozoa), but those with spermatozoa had significantly higher levels of serum inhibin
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TABLE 2 Data on individual men with idiopathic nonobstructive azoospermia in whom testicular sperm extraction was successful. Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
FSH level (IU/mL)
Inhibin B level (pg/mL)
29.75 32.2 46.3 28.2 32.3 31.9 20.6 19 33.9 12.1 1.9 13 35 15.5 21.5 16.45 20 36.7 52 16.76 3.5 20.7 9.76 2.8 30 23.3 9 6.4 18 2.7
ND ND 4.0 17.04 20.0 23.0 27.0 36.0 43.0 51.0 60.15 62.0 62.0 66.5 77.0 80.0 80.0 84.0 86.0 86.6 100.0 115.0 125.0 139.89 141.0 160.0 176.0 222.0 226.0 309.0
Note: ND ⫽ not detectable. Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
B than did those without spermatozoa (89.31 ⫾ 73.24 pg/mL vs. 19.23 ⫾ 22.34 pg/mL; P⫽.00009) (Tables 4 and 5).
To analyze further the diagnostic accuracy (predictive value of sperm retrieval) of inhibin B to discriminate between success and failure with TESE, the area under the ROC curves were determined for serum FSH and inhibin B (Fig. 1). The area under the ROC for inhibin B in predicting the likelihood of successful TESE was significantly higher than that for FSH. The ROC curve analysis was also used to determine the best threshold value for inhibin B in predicting success or failure of TESE. The inhibin B value that discriminated best between success and failure of TESE was 53 pg/mL (sensitivity, 91.11% [CI, 0.786 – 0.9764]; specificity, 75.86% [CI, 0.6276 – 0.8618]). Positive and negative predictive values were 0.7455 and 0.9167, respectively.
DISCUSSION Recent reports have suggested that recovery of testicular spermatozoa may be possible in more than 50% of men with nonobstructive azoospermia, regardless of clinical variables, size of the testes, or plasma FSH concentration (13–15). Fresh and cryopreserved testicular spermatozoa from azoospermic men have been used successfully in ICSI (16), which allows TESE to be performed before ovarian stimulation. Discovery of a reliable marker that predicts and correlates well with the presence of testicular spermatozoa on TESE in patients with nonobstructive azoospermia remains a high diagnostic priority. Follicle-stimulating hormone is currently regarded as the most important endocrine variable in evaluation of male testicular function (17). Its secretion is suppressed by the testicular hormone inhibin, which is produced in Sertoli cells and is therefore a serum marker for Sertoli cell function. Castration results in undetectable inhibin B levels, indicating that circulating inhibin B is produced by the testes (4). Nevertheless, deKretser et al. (18) postulated that inhibin levels did not differ in fertile controls and subfertile men with testicular disorders; this finding may have resulted from
TABLE 3 Serum FSH and inhibin B values. Group Idiopathic nonobstructive azoospermia (I) Nonobstructive azoospermia of identifiable (II) Obstructive azoospermia (III) Fertile volunteers (IV)
No. of patients
Serum FSH level (IU/mL)
Serum inhibin B level (pg/mL)
72 6 15 10
20.15 ⫾ 11.42a 36.13 ⫾ 9.66b 3.95 ⫾ 1.63c 7.37 ⫾ 8.18
48.43 ⫾ 60.74d 13.33 ⫾ 24.91e 156.83 ⫾ 80.14f 231.20 ⫾ 114.62
Note: Values are means ⫾ SD. a Group I vs. group II, P⫽.041; group I vs. group III, P⬍.0005; group I vs. group IV, P⫽.004. b Group II vs. group III, P⫽.002; group II vs. group IV, P⫽.001. c Group III vs. group IV, P⫽0.725. d Group I vs. group II, P⫽.086; group I vs. group III, P⫽.001; group I vs. group IV, P⫽.003. e Group II vs. group III, P⬍.0005; group II vs. group IV, P⫽.001. f Group III vs. group IV, P⫽.416. Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
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TABLE 4 Serum FSH and inhibin B values in men with idiopathic nonobstructive azoospermia. Group Idiopathic nonobstructive azoospermia with successful TESE with failed TESE
No. of patients (%)
Serum FSH level (IU/mL)
Serum inhibin B level (pg/mL)
72 (100) 30 (41.7) 42 (58.3)
20.15 ⫾ 11.42 21.37 ⫾ 12.92a 19.27 ⫾ 10.28a
48.43 ⫾ 60.74 89.31 ⫾ 73.24b 19.23 ⫾ 22.34b
Note: Values are means ⫾ SD. TESE ⫽ testicular sperm extraction. a Successful TESE vs. failed TESE, P⫽.97. b Successful TESE vs. failed TESE, P⫽0.00009. Brugo-Olmedo. Inhibin B and nonobstructive azoospermia. Fertil Steril 2001.
the low specificity of the inhibin assay used. The few reports on serum levels of inhibin B relative to quality of spermatogenesis (4, 5, 19 –21) seem to strongly indicate that inhibin B is an important marker of the competence of Sertoli cells and of the presence of spermatogenesis in the human. Pierik et al. (21) found a significant correlation between inhibin B levels and sperm concentration, sperm count, and testicular volume. They provided strong evidence that inhibin B is a valuable marker of the presence of spermatogenesis by showing a significant positive correlation between inhibin B levels and the most accurate assessment of spermatogenesis in their setting, the testicular biopsy score. Few reports indicated that serum inhibin B level is a predictor of the presence of testicular spermatozoa in azoospermic men, and results have been conflicting (9, 10). We compared serum FSH and inhibin B level in positive controls (fertile males with normozoospermia and patients with obstructive azoospermia) and men with idiopathic nonobstructive azoospermia. We also studied these variables in subgroups of nonobstructive azoospermic men with or without spermatozoa on TESE.
Patients with spermatozoa on biopsy have undeniable evidence of focal spermatogenesis; however, men with negative findings may still have areas of focal spermatogenesis that could have been missed on biopsy. Therefore, use of TESE results as the end point and the gold standard to be correlated with serum FSH or inhibin B levels as predictive markers is somewhat unreliable. Prediction of the presence or absence of germinal epithelium in abnormal testes from azoospermic patients is of utmost importance in planning treatment. If the couple decides to proceed with the attempt despite the available in-
FIGURE 1 Receiver-operating characteristic curves of inhibin B and FSH in discriminating success or failure of testicular sperm extraction in men with idiopathic nonobstructive azoospermia.
TABLE 5 Percentage of men with idiopathic nonobstructive azoospermia according to FSH and inhibin B cut-off levels. FSH serum level Good prognosis: FSH ⬍ 19 No. of patients: 38 13 (34.2%); successful TESE 25 (65.8%); failed TESE
Bad prognosis: FSH ⱖ 19 No. of patients: 34 17 (50%); successful TESE 17 (50%); failed TESE
Inhibin B serum level Good prognosis: IB ⬎ 53 No. of patients: 24 20 (83.3%); successful TESE 4 (16.7%); failed TESE
Bad prognosis: IB ⬍ 53 No. of patients: 48 10 (20.8%); successful TESE 38 (79.2%); failed TESE
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formation, biopsy can be performed before the stimulation cycle in the female partner is started; thereby sparing her a possibly unnecessary procedure. If sperm is found unexpectedly in the testicular tissue, it can be frozen for ICSI in the future, avoiding a second biopsy in the male partner. The accuracy of the basal serum inhibin B level in predicting presence or absence of germinal epithelium in patients with nonobstructive azoospermia must be evaluated further in a larger sample. Individual serum levels of inhibin B serum cannot currently be used as the definitive test for evaluating the presence or absence of spermatozoa in testicles of men with nonobstructive azoospermia; in these patients, TESE remains the most accurate diagnostic method.
8. 9.
10.
11.
12. 13. 14.
Acknowledgments: The authors thank L. Vargas, M.D., M. Albamonte, M.D., and V. Bertollino, M.D. for help with data collection and Ms. N. Durand, our librarian, for professional help.
15.
16.
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