Ultrasound, antisperm antibody, and hormone profiles after testicular Trucut biopsy

FERTILITY AND STERILITY VOL. 75, NO. 2, FEBRUARY 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Ultrasound, antisperm antibody, and hormone profiles after testicular Trucut biopsy E. Kristine Steele, B.Sc., M.D., M.R.C.O.G., a Peter K. Ellis, M.R.C.P., F.R.C.R., b Sheena E.M. Lewis, Ph.D., a and Neil McClure, M.D., M.R.C.O.G. a,c The Queen s University of Belfast, Institute of Clinical Science, Belfast, Northern Ireland, United Kingdom Received May 10, 2000; revised and accepted August 3, 2000. Financially supported by the Department of Health and Social Services and Fertility Research Trust, Northern Ireland, United Kingdom. Reprint requests: E. Kristine Steele, M.D., School of Medicine, Obstetrics and Gynecology, Queen s University Belfast, Grosvenor Road, Belfast BT12 6BJ, Northern Ireland, United Kingdom (FAX: 44-28-90-248005; E-mail: n.mcclure@qub. ac.uk). a Department of Obstetrics and Gynecology. b Department of Radiology, Royal Group of Hospitals Trust, Grosvenor Road, Belfast, Northern Ireland, United Kingdom. c Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. 0015-0282/01/$20.00 PII S0015-0282(00)01700-3 Objective: To assess the effect of Trucut needle biopsy on the ultrasound appearances of the testis in obstructive and nonobstructive azoospermia to test serum samples for antisperm antibodies and gonadotropin and testosterone levels. Design: Prospective case analysis. Setting: IVF unit. Patient(s): Sixteen subjects with obstructive azoospermia had postbiopsy ultrasound scans, 18 had assessment of hormone profiles, and 20 had evaluation of antisperm antibodies. Intervention(s): Trucut needle testicular biopsies under local anesthetic. Main Outcome Measure(s): Postbiopsy testicular ultrasound, the presence of serum antisperm antibodies, and follicle stimulating and luteinizing hormone and testosterone levels. Result(s): There were no postbiopsy hematomas or scars, antisperm antibodies did not develop, and pituitary gonadotropins did not rise nor testosterone levels fall. Conclusion(s): Trucut needle testicular biopsy in men with obstructive azoospermia is not associated with defects of parenchymal structure or function. (Fertil Steril 2001;75:423 8. 2001 by American Society for Reproductive Medicine.) Key Words: Trucut needle, ultrasound, antisperm antibodies, hormone profiles Intracytoplasmic injection of sperm aspirated from the testis by open or needle biopsy has offered fertility even to men with azoospermia. However, there are concerns about the safety of these procedures. The distribution of the vascular supply to the testis makes any sort of surgical penetration of the tunica albuginea hazardous, irrespective of the underlying testicular pathology. The testicular arterial supply penetrates the tunica and travels under its surface in a circumferential fashion, sending branches down each of the connective tissue septae that divide the testis into lobules (1). The subtunical arteries are end arteries, and damage to one could lead to devascularization of an area of parenchyma. No one area of the tunica can be blindly penetrated without the potential for damage to a vessel (2). Sixty percent of subjects with nonobstructive azoospermia will produce small numbers of sperm in tiny foci of normal spermatogenesis (3). To improve the success of sperm retrieval by testicular biopsy, some advocate the excision of large pieces of tissue (3), whereas others have suggested that multiple random samples should be obtained with a fine needle to biopsy, one hopes, one of these foci (4). Friedler et al. (4) compared the success rates of these two procedures by taking three large, open biopsies after making six separate entries with a fine needle. They found the fine-needle technique to be less successful than open biopsy at retrieving sperm, presumably because of the smaller total volume sampled. Schlegel and Su (1) performed testicular ultrasonography on subjects with nonobstructive azoospermia 3 months after open biopsy. Two subjects developed testicular atrophy, and 82% of subjects had hypoechoic areas suggestive of resolving hematomas. At follow-up at 6 423

months, most of these had resolved, leaving linear scars. These authors recommended avoiding multiple incisions in the tunica, minimizing repeated testicular extraction attempts, and further, speculated that multiple blind fine-needle procedures would not have any less risk of vascular injury than open biopsies. Ron-El et al. (5) found that 64% of a group of men with nonobstructive azoospermia had hyperechoic areas consistent with linear scars after substantial open biopsies. They also agreed that open and fineneedle biopsies could have the same potential for vascular injury. However, none of the six obstructive azoospermic subjects examined had any ultrasound lesions on scanning after fine-needle biopsy. Manning et al. (6) studied the effects of large open testicular biopsies on the hormone profiles of men with nonobstructive azoospermia, all of whom had normal testosterone concentrations before biopsy. However, testosterone levels fell significantly in all subjects 6 months after testicular sperm extraction, just over half of them to below the lower limit of normal. Although the levels had risen again by 12 months, they were not back to their original values. The fall in levels was more marked in patients who had had the most biopsies performed and in those in whom no spermatozoa were retrieved. Manning et al. therefore recommended the cryopreservation of testicular tissue in men with nonobstructive azoospermia to avoid the necessity of repeated biopsies. By contrast, no such studies have been reported in men with obstructive azoospermia. Because open biopsies in these cases do not involve the removal of such a high percentage of the testicular tissue, the hormone profile would be expected to be less affected. However, a large area of hematoma or complete testicular atrophy after the biopsy might have significant long-term consequences. Antisperm antibodies can be found in the fertile and infertile man and in the woman (7). Sperm are shielded from exposure to the immune system by the blood testis barrier. Although breach of this barrier from trauma, surgery or infection results in the formation of antisperm antibodies, in many instances, there is no such history. Antibodies are formed to antigens on the surface of sperm. However, the majority of sperm surface antigens do not regulate important functions; therefore, antibodies against these antigens do not have a significant effect on fertility except by clumping of sperm and decreasing motility and the ability to fertilize oocytes spontaneously. Identifying antisperm antibodies in seminal plasma is not relevant in men with obstructive azoospermia, and their presence must be sought in serum. Antisperm antibodies can be measured in several ways. Sperm agglutination tests, ELISA, mixed antiglobulin reaction (MAR), and immunobead testing (8) are the most common. The MAR test is based on the Coomb s test from hematology. Sensitized group O Rhesus-positive erythrocytes are mixed with the sperm and with a specific antihuman IgG antiserum. If the sperm carry IgG on their surface, a mixed-agglutination reaction occurs (9). In this study, we aim to determine the effects of Trucut biopsy on the ultrasound appearances of the testis and the hormone profiles of subjects with azoospermia. Further, the incidence of antisperm antibodies after Trucut biopsy is also reported. MATERIALS AND METHODS Subjects with obstructive and nonobstructive azoospermia were recruited from the Regional Fertility Center, Belfast. The two groups were differentiated by testicular volume and serum FSH and LH levels. Ethical approval was obtained from the Queen s University of Belfast Research and Ethics Committee (Institutional Review Board approval was not sought because it is not deemed necessary by the university). Anesthetic and Testicular Biopsy Trucut testicular biopsies were performed as described elsewhere (10). Briefly, the testis was anesthetized by infiltrating around the spermatic cord with bupivacaine and the skin with direct lidocaine injection. The testicular biopsies were taken with a 14-gauge Trucut needle after the skin and tunica had been punctured with a scalpel. Testicular Ultrasound All subjects underwent bilateral testicular ultrasound before biopsy to determine baseline appearance. Scans were repeated 3 months after the biopsy. All scans were performed by one of us (P.K.E.). For scanning, the scrotum was elevated onto a towel draped over the subject s thighs. An ATL Ultramark 9 scanner (Advanced Technology Labs, Bothell, WA) was used with a 10-mHz small-parts probe to provide increased resolution of the scrotal contents. Images of both testes were obtained in sagittal and transverse planes. Hard-copy images were obtained of normal and abnormal features for further comparison and reporting. Normal testes have a homogeneous granular echo texture of uniformly distributed medium-level echoes (11). The mediastinum testis is an echogenic linear area containing the testicular vessels and ducts. It can be confused for a scar, but color Doppler will reveal flow in the vessels. The epididymis is slightly more echogenic than the testis and is situated posterolaterally. Epididymal cysts are the result of dilatation of the epididymal tubules and are seen in 20% 40% of scans (11). Hormone Profiles Hormone profiles were determined on the day of testicular biopsy and again at their next visit to the hospital after the biopsy (usually after 4 weeks). Serum FSH and LH were measured by time-resolved fluoroimmunoassay by the Auto- DELFIA technology using reagents supplied by EG&G Wallac, Turku, Finland. The interassay coefficient of variation 424 Steele et al. 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for both assays at all serum levels encountered was 4%. Serum testosterone was measured by radioimmunoassay by COAT-A-COUNT technology using reagents supplied by Diagnostics Products Corporation, Los Angeles. The interassay coefficient of variation at a mean value of 19.6 nmol/l was 6.4%. Serum Antisperm Antibody Assessment Blood was collected for antisperm antibody estimation from each subject after biopsy (after 4 weeks at their clinic review) and spun for serum storage. Subsequently, antisperm antibody assessment was carried out using the Indirect SpermMar Test (Conception Technologies, San Diego, CA). Samples were thawed at room temperature before being inactivated at 56 C for 45 minutes in a water bath. The inactivated serum was then serially diluted 1/16 with Biggers, Whitten, and Whittingham medium (BWW; see Reference (12). Antisperm antibody negative sperm were obtained and the concentration, after density centrifugation, adjusted to 20 10 6 /ml with BWW. Fifty microliters of inactivated serum was mixed with 50 L of donor sperm and incubated for 1 hour at 37 C. Ten microliters each of sperm serum solution, SpermMar antiserum, and IgG SpermMar latex particles were placed onto a microscope slide and thoroughly mixed. A coverslip was placed on the slide, and 100 motile sperm were assessed after 2 3 minutes for the number that had attached latex particles. If there were no sperm with attached latex particles, the slide was reexamined after 10 minutes to ensure that it was a true negative result. RESULTS Testicular Ultrasound Sixteen azoospermic subjects underwent prebiopsy and postbiopsy ultrasound scanning. Four other subjects who attended for prebiopsy scans did not attend for postbiopsy scans despite numerous reminders. Thirteen of the 16 had obstructive azoospermia and three, nonobstructive azoospermia. Six of the subjects with obstructive azoospermia had no identifiable cause for it; obstructive azoospermia in three was from vasectomy, in two from paraplegia, and in two from ejaculatory failure. All of the subjects with nonobstructive azoospermia had Sertoli Cell Only Syndrome. Prebiopsy Scans Fifteen sets of prebiopsy films were available for reporting. Eight of the prebiopsy scans were entirely normal. Although six of the remaining seven subjects testicular scans were also normal, all had minor abnormalities of the extratesticular tissues. Four had tiny unilateral epididymal cysts, one had a small hydrocele (left side), and the final subject had a small left-sided varicocele. The last subject s testes showed small bilateral focal calcifications. Postbiopsy Scans Sixteen scans were available for reporting (Table 1). There was no sign of testicular atrophy on any of the scans TABLE 1 Pre- and post-testicular biopsy ultrasound findings. Patient Diagnosis Pre biopsy scan Post biopsy scan 1 Obstructive Normal Normal 2 Obstructive Normal Normal 3 Obstructive Normal Normal 4 Obstructive Normal Normal 5 Vasectomy Normal Normal 6 Anejaculatory Normal Normal 7 Paraplegia Normal Normal 8 Sertoli only Normal Normal 9 Vasectomy Epididymal cysts Epididymal cysts 10 Vasectomy Epididymal cysts Epididymal cysts 11 Obstuctive Epididymal cysts Epididymal cysts 12 Sertoli only Epididymal cysts Epididymal cysts 13 Sertoli only Varicocele Varicocele 14 Paraplegia Testicular calcification Testicular calcification 15 Obstructive Hydrocele Normal 16 Obstructive Films missing Epididymal cysts on volumetric assessment, and none showed any evidence of resolving hematoma or scarring within the testicular parenchyma (Figures 1 and 2). The scan from the subject with intratesticular calcifications was unchanged. Six of the scans again showed abnormalities of the extratesticular tissues, five of which were identical to the prebiopsy findings (four with tiny unilateral epididymal cysts and one with a small varicocele). The subject with the small hydrocele had no sign of this on postbiopsy scanning. The final subject had a normal testicular scan with no scars or hematomas but with small bilateral epididymal cysts. This subject s prebiopsy scan was lost, so no comment could be made as to whether testicular atrophy had occurred, except that the postbiopsy testicle measured 4.5 2.5 cm, which is within the normal range. Hormone Profiles Eighteen subjects had blood sent for postbiopsy hormone profiles. For 14 of these subjects, prebiopsy hormone profile results were available for comparison. The four subjects with only postbiopsy hormone profiles all had levels within the normal ranges (FSH, 1.5 8.0 IU/L; LH, 1.5 9.0 IU/L; and testosterone, 12 nmol/l). In three prebiopsy and five postbiopsy samples, the testosterone results were unavailable. Sixteen of the subjects were known to have obstructive azoospermia on the basis of histologic evaluation of part of each of the biopsies that were reported as type A 1 azoospermia (13). Histology results were unavailable for the other two subjects, but one of them was azoospermic after a vasectomy, and therefore, histology should have been type A 1. The cause of the obstructive azoospermia was unknown in 10 subjects; of the others, in six, the obstructive azoospermia was due to vasectomy, one subject was paraplegic, and one had congenital bilateral absence of the vas deferens. FERTILITY & STERILITY 425

FIGURE 1 Ultrasound appearances of both testes before biopsy and of the left testis after biopsy in a patient with obstructive azoospermia. TABLE 2 Pre- and post-biopsy hormone profile results. a FSH (1.5 8.0 IU/l) LH (1.5 9.0 IU/l) Testosterone ( 12 nmol/l) Patient Pre Post Pre Post Pre Post 1 4.1 3.8 2.5 2.1 9.9 11.8 2 3.6 3.1 4.8 3.9 3 2.2 2.3 2.1 2.1 15.2 9.3 4 2 2.3 3.2 2.9 13.2 18.1 5 5.8 5.8 3.3 2.7 16.5 16.6 6 4 3.4 19.3 17.1 7 5.5 5.8 4 2.8 23.4 20 8 8.1 10.3 2.4 3.6 9 2.2 2.9 3 3.5 10 4.1 5.3 3.8 2.6 22 8.5 8.8 3.7 2.4 15.5 14.5 12 9.5 10.4 6.1 6.3 13 10.5 11.5 3.7 5.5 14.7 12.6 14 9.9 4.8 a There was no significant difference between the original and repeat results (p 0.05; Wilcoxon matched pairs test). For the 14 subjects for whom paired FSH levels were available, the mean ( SD) prebiopsy level was 5.7 3.0, and the postbiopsy level was 5.8 3.2. There was no significant difference between these values (P.05; Wilcoxon matched-pairs test; Table 2). The mean ( SD) prebiopsy LH level was 3.6 1.1, and after biopsy, it was 3.4 1.3. There was no significant difference between these values (P.05; Wilcoxon matchedpairs test). For the nine subjects with matched testosterone levels prebiopsy and postbiopsy, for comparison, the mean ( SD) prebiopsy testosterone level was 15.9 4.0, and after biopsy, it was 15.0 3.6. There was no significant difference between these values (P.05; Wilcoxon matched-pairs test). Postbiopsy Antisperm Antibodies Results were available for 20 postbiopsy subjects. The cause of the azoospermia was unknown in 10 of these men because of paraplegia in two of them, because of vasectomy in six of them, and because of congenital absence of the vas deferens in two of them (CBAVD, one with respiratory cystic fibrosis). Results of histologic evaluation were unavailable for three of the subjects (two of these subjects definitely had obstructive azoospermia clinically). Sixteen of the samples were reported as azoospermia type A 1 (13), and one was reported as type C. Sixteen of the serum samples were negative for antisperm IgG, and four were positive. Of the four samples, 65%, 45%, 35% and 31% of the motile sperm respectively had latex particles adhering to the head, midpiece, or tail. Two of these 426 Steele et al. The post-trucut biopsy testis Vol. 75, No. 2, February 2001

FIGURE 2 Ultrasound appearances of both testes before biopsy and of the left testis after biopsy in a patient with nonobstructive azoospermia. men were azoospermic because of vasectomy (45% and 31%) and the other two, because of CBAVD, one with respiratory cystic fibrosis and the other a carrier of a common gene mutation. DISCUSSION Although there are published reports of the long-term sequelae of open testicular biopsy, the effects of Trucut needle testicular biopsy on testicular anatomy and function are unknown. We have now demonstrated that even though the Trucut technique provides large numbers of sperm, the long-term effects are negligible. No testicular scars or hematomas were observed in the subjects studied who had testicular sperm extraction by Trucut needle, and there was no evidence of testicular atrophy in any of the biopsied testes. This is in agreement with a previous report by Ron-El et al. (5) assessing the impact of needle sperm retrieval on the testes of men with obstructive azoospermia that found no focal lesions on scans 6 months later. However, in that study, five of these men only had a fine-needle epididymal sperm aspiration procedure carried out, and the final subject had bilateral testicular sperm aspiration carried out using a much smaller needle (21 gauge) than the Trucut (14 gauge). Most reports of postbiopsy ultrasonography have concentrated on subjects with nonobstructive azoospermia (1, 5). Both of these papers reported high incidences of hematomas and scars after open biopsies. However, these biopsies were described as being large or substantial to increase the chances of obtaining a few sperm. A substantial biopsy from a small soft testis in nonobstructive azoospermia will involve the removal of a greater percentage of the testicular substance than the same size of biopsy from a normal-sized testis in obstructive azoospermia. Schlegel and Su (1) recommended that large incisions and multiple incisions be avoided because of the risk of vascular injury. There is also a potential risk of vascular injury with blind-needle biopsy of the testis (5). The main advantage of using the large-bore Trucut needle over a fine needle is that one biopsy provides enough sperm for a fresh ICSI treatment cycle and sufficient sperm for cryopreservation for future cycles. In this way, the risk of blind vascular injury is reduced compared with the case of multiple fine-needle biopsies repeated for each treatment cycle. From our study, it would appear that Trucut needle biopsy is not associated with hematomas or scars, even in those with nonobstructive azoospermia. However, it must be emphasized that the number of subjects in this study was small and that further study, especially of those with nonobstructive azoospermia, is needed because previous reports have suggested that multiple needle biopsies may not be safe (5). The use of the Trucut needle may be suitable for further refinement, in nonobstructive azoospermia, by the addition FERTILITY & STERILITY 427

of color Doppler. Intratesticular blood flow is impaired in nonobstructive azoospermia, but areas of normal flow can be identified with color Doppler (14). When these areas were sampled with a fine needle, normal spermatogenesis was identified. If a Trucut needle were used to sample such areas combined with tubule milking, enough sperm for both fresh and freeze-thawed treatment cycles might be obtained, thereby avoiding the need for repeat biopsy (10). In this study, we identified no significant elevation in FSH and LH or reduction in post biopsy testosterone levels from any subject. These results are in keeping with the absence of testicular damage on post biopsy scans. The reports of a reduction in testosterone levels (6) after biopsy in nonobstructive azoospermia patients can be explained when one takes into account that 82% of subjects had resolving hematomas (1), and 64% of subjects had parenchymal scars (5). Damage or destruction of large areas of parenchyma would include Leydig cells and, thus, result in a drop in testosterone levels. A loss of seminiferous tubules would also result in a drop in the levels of inhibin and, therefore, in increased FSH released from the anterior pituitary. Of the 20 subjects from whom serum was studied for the presence of antisperm antibodies, there were four positive results. Six subjects were azoospermic because of vasectomy; of those, two were antibody positive. The connection between vasectomy and antisperm antibodies is well recognized (9). Congenital absence of the vas deferens in the presence of normal spermatogenesis (as was the case for the two remaining antibody-positive subjects) is often associated with antibody production because of the increased probability of sperm entering the lymphatic system and triggering the immune response (9). In conclusion, azoospermic subjects who undergo Trucut needle testicular biopsy do not appear to develop testicular damage, antisperm antibodies, or a significant alteration in hormone profiles as a result of the procedure. Further work is needed to confirm this in subjects with nonobstructive azoospermia. Acknowledgments: The authors thank Dr. Brian Sheridan of the Supraregional Endocrine Laboratory, Royal Group of Hospitals Trust for coordinating the hormone assays and Mr. Roy Creighton for converting the ultrasound films into photographs. The authors also thank Mrs. Dorothy White (secretary to P.K.E.) for booking all of the ultrasound scan appointments. References 1. Schlegel P, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod 1997;12:1688 92. 2. Jarow JP. Clinical significance of intratesticular arterial anatomy. J Anat 1991;145:777 9. 3. Silber SJ, Johnson L. Are spermatid injections of any clinical value? ROSNI and ROSI revisited. Hum Reprod 1998;13:509 23. 4. Friedler S, Raziel A, Strassburger D, Soffer Y, Komarovsky D, Ron-El R. Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum Reprod 1997;12:1488 93. 5. Ron-El R, Strauss S, Friedler S, Strassburger D, Komarovsky D, Raziel A. Serial sonography and colour flow Doppler imaging following testicular and epididymal sperm extraction. Hum Reprod 1998;13:3390 3. 6. Manning M, Junemann KP, Alken P. Decrease in testosterone blood concentrations after testicular sperm extraction for intracytoplasmic sperm injection in azoospermic men. Lancet 1998;352:37. 7. The ESHRE Capri Workshop Group. Male infertility update. Hum Reprod 1998;13:2025 32. 8. Zouari R, De Almeida M, Feneux D. Effect of sperm-associated antibodies on the dynamics of sperm movement and on the acrosome reaction of human spermatozoa. J Reprod Immunol 1992;22:59 72. 9. Matson PL. Sperm antibodies: formation and significance. In: Yovich JL, Yovich JGG, eds. Gametes the spermatozoon. Cambridge, UK: Cambridge Academic Press, 1995:237 49. 10. Steele EK, McClure N, Maxwell RJ, Lewis SEM. A comparison of DNA damage in testicular and proximal epididymal spermatozoa in obstructive azoospermia. Mol Hum Reprod 1999;5:831 5. 11. Dambro TJ, Stewart RR, Carroll BA. The scrotum. In: Rumack CM, Wilson SR, Charboneau JW, eds. Diagnostic ultrasound. Vol. 1. St. Louis, MO: Mosby Year Book Inc, 1998:791 821. 12. Biggers JD, Whitten WK, Whittingham DG. The culture of mouse embryos in vitro. In: Methods in mammalian embryology. San Francisco: Freeman Press, 1971. 13. Dajani YF. Testicular biopsy: an update. Curr Diagn Pathol 1998;5: 17 22. 14. Foresta C, Garolla A, Bettella A, Ferlin A, Rossato M, Candiani F. Doppler ultrasound of the testis in azoospermic subjects as a parameter of testicular function. Hum Reprod 1998;13:3090 3. 428 Steele et al. The post-trucut biopsy testis Vol. 75, No. 2, February 2001