FERTILITY AND STERILITY VOL. 72, NO. 3, SEPTEMBER 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. MALE FACTOR Identification of seminiferous tubule aberrations and a low incidence of testicular microliths associated with the development of azoospermia Gary D. Smith, Ph.D.,* Ian Steele, Ph.D., Randall B. Barnes, M.D.,* and Laurence A. Levine, M.D. The University of Chicago, and Rush-Presbyterian St. Luke s Medical Center, Chicago, Illinois Received July 14, 1998; revised and accepted March 15, 1999. Reprint requests and present address: Gary D. Smith, Ph.D., Department of Obstetrics and Gynecology, University of Michigan, F4826 Mott Hospital, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0272 (FAX: 734-936-8617; E-mail: smithgd@umich.edu). * Department of Obstetrics and Gynecology, The University of Chicago. Department of Geophysical Sciences, The University of Chicago. Department of Urology, Rush-Presbyterian St. Luke s Medical Center. 0015-0282/99/$20.00 PII S0015-0282(99)00271-X Objective: To evaluate the use of percutaneous testicular sperm aspiration in the assessment of azoospermia and its association with seminiferous tubule microliths. Design: Case report. Setting: Tertiary care fertility center in a university hospital. Patient(s): Male undergoing infertility evaluation. Intervention(s): Testicular biopsy and percutaneous testicular aspiration. Main Outcome Measure(s): Serum hormone analysis, sperm concentration in semen, spermatogenesis in samples from testicular biopsies and aspirations, and microlith composition. Result(s): A patient presented for infertility evaluation with a history of severe oligospermia that progressed to azoospermia. The serum testosterone concentration (357 ng/dl) and LH concentration (9.2 miu/ml) were normal and the serum FSH concentration (18.3 miu/ml) was elevated. Testicular biopsy results indicated spermatogenic hypoplasia with limited spermatozoa. Seminiferous tubules obtained by percutaneous testicular aspiration were structurally aberrant, with multiple diverticula. Microliths averaging 120 m in diameter were observed within and blocking the seminiferous tubules. The microliths were composed of calcium phosphate (hydroxyapatite) in both the core and peripheral regions. Electron microscopy revealed a high degree of collagen-like material within the peripheral zone. Conclusion(s): The presence of seminiferous tubule microliths is associated with the development of azoospermia. In patients with a low incidence of seminiferous tubule microliths and aberrant seminiferous tubule architecture, percutaneous testicular aspiration may provide a diagnostic advantage over testicular biopsy. (Fertil Steril 1999;72:467 71. 1999 by American Society for Reproductive Medicine.) Key Words: Percutaneous testicular sperm aspiration, testicular biopsy, focal spermatogenesis, testicular microliths Calcified concretions, or microliths, located in the seminiferous tubules are rare and more commonly observed in children (1) than adults (2, 3). Testicular microliths have been reported to be associated with cryptorchidism (1, 4), Klinefelter syndrome (5), testicular dysgenesis (6), and testicular tumors (7). The mechanism of testicular microlith formation is unclear, with reports suggesting both intratubular (8, 9) and extratubular (10) origins. The abundance of microliths within the testes can range from infrequent to involving a significant number of seminiferous tubules; the condition is termed testicular microlithiasis, and it can be detected with radiologic (11) or sonographic (12) techniques. The occurrence and detection of a low incidence of testicular microliths may be misrepresented because of inherent spatial limitations in the current method of detection, testicular biopsy. The relation of a low incidence of testicular microliths, or testicular microlithiasis, and infertility remains to be elucidated. Schantz and Milsten (2) reported a case involving infertility associated with testicular microlithiasis in which 30% 40% of the seminiferous tubules, as assessed by testicular biopsy, contained microliths. In this report, we describe the use of 467
testicular biopsy and percutaneous testicular aspiration to identify a low incidence of testicular microliths in an individual with azoospermia. In addition, we conducted studies to elucidate seminiferous tubule histology and morphology, and microlith composition. MATERIALS AND METHODS Patient A 33-year-old man and his wife presented for evaluation of infertility at The University of Chicago Hospitals. The woman had no apparent cause of infertility. Over a 10-month period, three semen analyses were performed; the standards of normality used were a sperm concentration of 20 10 6 /ml, motility of 50%, and morphology of 30% normal forms (13). Serum samples were obtained for evaluation of FSH concentrations (normal 0.9 15 miu/ml), LH concentrations (normal 1.3 12.9 miu/ml), prolactin concentrations (normal 2 18 ng/ml), and total testosterone concentrations (normal 194 833 ng/dl). Open testicular biopsy was performed for histologic evaluation of spermatogenesis. Institutional review board approval was not obtained because observational data were acquired during normal therapeutic interventions for the treatment of infertility. Testicular Biopsy and Percutaneous Sperm Aspiration A bilateral open testicular biopsy was performed as previously described (14). Testicular tissues were fixed in 10% formaldehyde, embedded in paraffin, subjected to serial sectioning at a thickness of 6 m, and stained with hematoxylin and eosin. Bilateral percutaneous testicular sperm aspirations were performed as previously described (15). In brief, under IV sedation and local anesthesia, a 19-gauge butterfly needle was inserted into the anterior midpole of the immobilized testicle. Suction was applied by pulling the plunger of a 30-mL syringe and was maintained by clamping the butterfly tubing. The needle was quickly advanced and retracted several times into multiple testicular regions without complete removal from the testicle until fluid and tissue were visualized within the butterfly tubing. Testicular aspirates were flushed from the tubing into a sterile test tube with a tuberculin syringe containing HEPESbuffered human tubal fluid medium (16). The contents of the testicular aspirates, which were comprised of predominantly seminiferous tubules, were evaluated under a stereomicroscope for overall morphology and evidence of spermatogenesis. Individual seminiferous tubules were dissected microscopically with 23-gauge needles to isolate material within the seminiferous tubules (i.e., seminiferous epithelium, germ cells, microliths). Microlith Physical Analysis For transmission electron microscope evaluation of testicular microlith composition, individual microliths were fixed in 2% paraformaldehyde/2% glutaraldehyde, rinsed in phosphate-buffered saline, and washed 4 6 times in 0.1M Na cacodylate buffer (ph 7.4) for 5 minutes each wash. One percent osmium tetroxide was used to postfix the microliths for 1 hour at room temperature until the specimens turned dark brown. Distilled water was used in five washes to rinse away any excess osmium. Fixed samples then were stained overnight in a 0.5% solution of uranyl acetate at 4 C in the dark. The stained samples were dehydrated through a graded series of ethanol solutions (35% 95%) for 5 minutes at each step. To ensure complete dehydration, the samples were rinsed in 100% absolute ethanol for a total of 30 minutes. The samples were infiltrated with a mixture of 100% ethanol:epon 812 (Electron Microscopy Sciences, Fort Washington, PA) for 30 minutes, subjected to several changes in total resin for 1 hour, and left in total resin overnight. The following day, the microliths were embedded in fresh Epon 812 and polymerized overnight at 65 C. The resulting blocks were pretrimmed and pale gold sections were cut using a Reichert- Jung D Ultramicrotome (Lieca Inc., Deerfield, IL). The sections were contrasted further with 1.5% uranyl acetate and lead citrate, for 5 minutes each, and viewed on a JEOL 100-CX II electron microscope (Peabody, MA) operated at 60 kv. The testicular microliths were coated with approximately 200 angstrom of carbon for conduction and then visualized and photographed using secondary electron images from an SX-50 electron microprobe (CAMECA, Paris, France). Isolated microliths also were mounted in epoxy and polished using a 1- m diamond to expose a surface through the near center. This polished surface was carbon-coated for conductivity and inserted into the sample chamber of the microprobe, then visualized and photographed using backscattered electron images. Qualitative energy-dispersive x-ray spectra were obtained from both the nucleus and the shell to gain insight into the composition of the layers. In addition, the microliths were mounted on the tip of a glass fiber using epoxy and an x-ray pattern was obtained using a 57.3-mmdiameter Gandolfi camera (Blake Industries, Inc., Scotch Plains, NJ) and Mn-filtered Fe K-alpha radiation with a 24-hour exposure. RESULTS On physical examination, the patient was tall, well developed, and moderately obese without signs of gynecomastia. The testicles were bilaterally descended and below average size, with an approximate volume of 5 ml. Bilateral epididymides and vas deferens appeared normal by palpation. Three consecutive semen analyses revealed normal volume, normal motility, ph of 8.0 8.5, morphology of 5% 6% normal forms (13), and a progressive decline in sperm concentration from oligozoospermia to azoospermia. Unfortunately, previous information regarding semen and 468 Smith et al. Testicular microliths and azoospermia Vol. 72, No. 3, September 1999
FIGURE 1 Composite micrograph of aberrant seminiferous tubules, tubule obstructions, and testicular microliths in a patient with acute-onset azoospermia. (A to D), Stereomicroscopic evaluation of seminiferous tubules obtained by percutaneous testicular aspiration. (A), Evidence of aberrant tubule morphology comprised of multiple diverticula and the presence of microliths. (B and D), Microliths composed of two concretions and a single encapsulation. The asterisk denotes the region of intratubule cellular buildup. (C), A single microlith that has dislodged from its location of obstruction on manual manipulation (arrow). Note the enlarged tubular pouch that contains intratubular debris, including germ cells. (E and F), Histologic representation of testicular microliths after testicular biopsy, tissue sectioning, and hematoxylin-and-eosin staining. Magnification, 40 (E) and 100 (F). (G), Scanning electron micrograph of an isolated testicular microlith approximately 140 m in diameter (bar 100 m). (H), Visualization of the multilayered composition of a testicular microlith sectioned through its center and then subjected to diamond polishing and microprobe analysis (bar 50 m). (I and J), Transmission electron micrographs depicting the microlith s fibrous collagen-like exterior encasing the acellular core region. Smith. Seminiferous tubule aberrations. Fertil Steril 1999. sperm characteristics was not available. The serum LH prolactin, and testosterone concentrations were within the normal ranges (9.2 miu/ml, 7 ng/ml, and 284 ng/dl, respectively). The serum FSH concentration was elevated (18.3 miu/ ml), indicative of a primary parenchymal abnormality. After the reduction in seminal sperm density from oligozoospermia to azoospermia, an open testicular biopsy was performed to evaluate spermatogenesis. The right testicular biopsy revealed rare seminiferous tubules with Sertoli cells, spermatogonia, primary and secondary spermatocytes, sper- FERTILITY & STERILITY 469
matids, and rare spermatozoa. Most of the tubules contained only Sertoli cells, consistent with germ cell aplasia. The left testicular biopsy revealed complete spermatogenesis with spermatozoa in approximately 40% of the tubules, whereas the rest of the tubules contained only Sertoli cells. In addition, both samples showed Leydig cells and tubular basement membranes of normal thickness. One to three individual microliths were apparent in samples from both testicles (Fig. 1E and F). On light microscopy, these microliths appeared to be multilayered, with a fibrous capsule. A subsequent percutaneous testicular aspiration was performed to attempt retrieval of spermatozoa for an assisted reproductive procedure. Gross microscopic visualization of aspirated seminiferous tubules revealed structural aberrations in seminiferous tubule morphology characterized by the presence of multiple tubular diverticula (Fig. 1A). Threedimensional examination of multiple seminiferous tubules enabled the identification of multiple, albeit low incidence, intratubule microliths (Fig. 1A D). Although most of these microliths were singular concretions (Fig. 1C), some appeared to be multiple concretions within a single capsule (Fig. 1A, B, and D). Of possible greater importance was the observation of tubule blockage by microliths and the accumulation of intratubule contents on one side of the microlith. In the most extreme examples, this blockage formed tubule enlargements that contained an extensive amount of cellular debris, including spermatozoa (Fig. 1C). To further assess the composition of testicular microliths, individual concretions were isolated from seminiferous tubules and evaluated with x-ray diffraction, qualitative electron microprobe analysis, and transmission electron microscopy. Isolated testicular microliths averaged 120 m in diameter. Scanning electron microscopy revealed a nearspherical form and a slightly undulating surface (Fig. 1G). A backscattered electron image of testicular microliths (Fig. 1H) obtained from the polished section clearly showed an internal structure consisting of a central round, but slightly off-center, core and an outer 20- m-thick shell composed of individual grains separated by dark intergranular areas. These individual grains appeared to form a layered structure within the shell but with a continuous layer at the surface. In the microprobe vacuum, the core region showed a continuous evolution of volatiles as seen by raising of the thin carbon coat over this portion. The origin of this is not known, but possibilities include vacuum drying of organic material, removal of polishing oil, and original body fluids trapped by the vacuum. This effect was not observed in the outer shell and was consistent with the distinctly different texture. To identify the constitution of the microliths, x-ray diffraction was performed and resulted in a film that showed seven slightly diffuse lines; the observed interatomic d- spacing is given in Table 1. This pattern matched well with the ASTM 9-432 pattern, suggesting that the crystalline TABLE 1 Interatomic d-spacing values for testicular microliths and hydroxyapatite. Testicular microliths observed interatomic d-spacing (angstrom) material in the sphere was hydroxyapatite, or Ca 5 (PO 4 ) 3 (OH). Qualitative energy-dispersive x-ray spectra were obtained from both the core and the shell and showed major Ca and P and minor Mg consistent with hydroxyapatite, with the minor Mg probably substituting for Ca in the structure. No chemical difference was noted between the core and the shell, although the x-ray data cannot indicate whether there was a difference in crystallinity between the apatite in the shell and that in the nucleus corresponding with the textural differences observed in the backscattered electron image (Fig. 1H). Transmission electron microscopy revealed that core regions of the microliths were acellular, consistent with a hydroxyapatite calcification (Fig. 1I). The external shell portion appeared to be collagen or collagen-like in conformation, with an acellular composition (Fig. 1I). This is the first ultrastructural characterization of testicular microliths, which appear to be similar to intrarenal microliths (17 19). DISCUSSION Hydroxyapatite ASTM 9-432 interatomic d- spacing (angstrom) 3.431 3.44 3.101 3.08 2.792 2.814 and 2.778 2.292 2.296 and 2.262 1.943 1.943 1.849 1.841 1.723 1.722 Smith. Seminiferous tubule aberrations. Fertil Steril 1999. Whereas most reports in the literature addressing testicular microliths have dealt with microlithiasis, in which numerous concretions reside in the seminiferous tubules, in this report we described the presence and ultrastructural composition of relatively infrequent microliths. The identification and incidence of infrequent testicular concretions may be misrepresented because of the use of testicular biopsy, which intrinsically provides fewer observational opportunities compared with microscopic analysis of seminiferous tubules recovered by percutaneous testicular aspiration. Stereomicroscopic observations of seminiferous tubules obtained by percutaneous testicular aspiration provided us with a threedimensional view of tubule structure and an opportunity to make observations on tubule morphology. These structural abnormalities were not, and cannot, be recognized from the two-dimensional views provided by testicular sections obtained from open testicular biopsy. 470 Smith et al. 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The question arises as to the frequency of structural seminiferous tubule aberrations in relation to severely compromised spermatogenesis and azoospermia. Because testicular biopsy has been the primary diagnostic procedure for evaluating spermatogenesis, this query remains to be elucidated. Whether these tubule abnormalities were congenital or acquired is unknown but may be important considering the possible heredity of seminiferous tubule abnormalities and the reproductive success of offspring. The possible transmission of genetic diseases or reproductive dysfunction, which could include reproductive structural abnormalities, has been reviewed (20). It must be considered that techniques such as testicular sperm aspiration or extraction coupled with intracytoplasmic sperm injection or round spermatid nucleus injection could perpetuate the propagation of these disorders. The origin of the observed testicular microliths is still unclear. It has been suggested that testicular microliths originate from the tunica propria and enter the seminiferous tubule by growth and compression (10). Although this may be the case, we did not identify any concretions in extratubular areas. The relation of testicular microliths and infertility has been an area of debate. An association between testicular microlithiasis, in which 30% 40% of the seminiferous tubules contain microliths, and male factor infertility has been reported (2). However, this is the first report of a patient with a low incidence of testicular microliths in whom tubular occlusion was identified in association with the development of azoospermia. It has been suggested that microliths may form seminiferous tubule obstructions in patients with below-normal tubular diameters (1). In this report, we demonstrated that blockage of seminiferous tubules by microliths results in obstruction of intratubule flow, as evidenced by a buildup of cellular debris behind microliths. Most spermatozoa isolated from these enlargements were amorphous, with a high degree of head abnormalities, including vacuolization. Whether these head abnormalities were present in the spermatozoa at the time of spermiation or were the result of a hostile intratubule environment resulting from the blockage of seminiferous tubules is currently unknown and requires further investigation. In addition, it is important to recognize that these microliths may be a consequence of a degenerative condition within the seminiferous tubules that itself leads to azoospermia. In conclusion, we demonstrated the usefulness of percutaneous testicular aspiration in obtaining diagnostic information regarding the structural morphology of seminiferous tubules and the presence of a low incidence of testicular microliths. These microliths can cause blockage of the seminiferous tubules; however, whether these microliths are a cause or a consequence of progression from oligospermia to azoospermia requires further investigation. Acknowledgments: The authors thank Carrie Cosola-Smith, D.V.M., M.D., for critical review of the manuscript and Edward K. Williamson, Ph.D., M.D., and The University of Chicago Cancer Center Electron Microscopy Facility for technical assistance in conducting transmission electron microscopy. References 1. Nistal M, Paniagua R, Diez-Pardo JA. Testicular microlithiasis in two children with bilateral cryptorchidism. J Urol 1979;121:535 7. 2. Schantz A, Milsten R. Testicular microlithiasis with sterility. Fertil Steril 1976;27:801 5. 3. Hobarth K, Susani M, Szabo N, Kratzik C. Incidence of testicular microlithiasis. Uroradiology 1992;40:464 7. 4. Sohval AR. Histopathology of cryptorchidism: a study based upon the comparative histology of retained and scrotal testes from birth to maturity. Am J Med 1954;16:346 51. 5. Lanman JT, Sklarin BS, Cooper HL, Hirschorn K. Klinefelter s syndrome in a ten-month-old mongolian idiot, report of a case with chromosome analysis. N Engl J Med 1960;263:887 91. 6. Turner JH, Bloodworth JMB. The testis. In: Bloodworth JMB, editor. Endocrine pathology. Baltimore: Williams & Wilkins, 1966:430. 7. Ikinger U, et al. Microcalcifications in testicular malignancy. Urology 1982;5:525 33. 8. Bunge RG, Bradbury JT. Intratubular bodies of the human testis. J Urol 1961;85:306 10. 9. Weinberg AG, Curraino G, Stone IC. Testicular microlithiasis. Arch Pathol 1973;95:312 4. 10. Nistal M, Martinez-Garcia C, Paniagua R. The origin of testicular microliths. Int J Androl 1995;18:221 9. 11. Smith SW, Brammer HM, Henry N, Frazier H. Testicular microlithiasis: sonographic features with pathologic correlation. Am J Roentgenol 1991;157:1003 4. 12. Doherty FJ, Mullins TL, Sant BR, Drinkwater MA, Ucci A. Testicular microlithiasis: a unique sonographic appearance. J Ultrasound Med 1987;6:389 92. 13. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, 1993. 14. Goldstein M. Surgical management of male infertility and other scrotal disorders. In: Walsh PC, Retik AB, Vaugh ED, Wein AJ, eds. Campbell s urology. 7th ed. Philadelphia: WB Saunders Co., 1998:1331 7. 15. Levine LA, Lisek EW. Successful sperm retrieval by percutaneous epididymal and testicular sperm aspiration. J Urol 1998;159:437 40. 16. Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubule fluid. Fertil Steril 1985;44:493 8. 17. Verbueken AH, Van Grieken RE, Verpooten GA, De Broe ME, Wedeen RP. Laser microprobe mass spectrometric identification of cyclosporin-induced intrarenal microliths in rat. Biological Mass Spectrometry 1992;21:590 6. 18. Boeve ER, Ketelaars GAM, Vermeij M, Cao LC, Schroder FH, De Bruijn WC. An ultrastructural study of experimentally induced microliths in rat proximal and distal tubules. J Urol 1993;149:893 9. 19. De Bruijn WC, Ketelaars GAM, Boeve ER, Sorber CWJ, Cao LC, Schroder FH. Electron energy-loss spectroscopical and image analysis of experimentally induced rat microliths II. J Urol 1993;149:900 5. 20. Cummins JM, Jequier AM. Concerns and recommendations for intracytoplasmic sperm injection (ICSI) treatment. Hum Reprod 1995;10: 1 8. FERTILITY & STERILITY 471