Urologia Internationalis Original Paper Urol Int 2004;73:23 27 DOI: 10.1159/000078799 Received: July 16, 2003 Accepted after revision: October 29, 2003 Fine-Needle Aspiration Cytology of the Testis: Can It Be a Single Diagnostic Modality in Azoospermia? a a b c a A. Srivastava M. Raghavendran M. Jain S. Gupta H. Chaudhary Departments of a Urology and Renal Transplantation, b Pathology, and c Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India Key Words Fine-needle aspiration cytology Follicle-stimulating hormone Intra-cytoplasmic sperm injection In vitro fertilization Sertoli cell only syndrome Vasoepididymal anastomoses Hypospermatogenesis Maturation arrest Testicular biopsy Azoospermia Abstract Purpose: The aim of this study was to determine whether fine-needle aspiration cytology (FNAC) of testis alone is sufficient to diagnose testicular function and whether follicle-stimulating hormone (FSH) estimation can be safely eliminated from the evaluation protocol of the azoospermic subject. Materials and Methods: We studied 46 adult azoospermic males who were infertile for more than 2 years following marriage. Hormonal profile was done in all. Later all 46 patients were subjected to bilateral FNAC of the testes. The cytological findings were correlated with histological findings. Results: We found 95.65% agreement between FNAC and testicular biopsy. Though serum FSH estimation was done in all patients in this series, in none of the cases did it affect overall management. Conclusion: FNAC is a quick, safe and minimally invasive modality. Following a well-performed semen analysis in an azoospermic subject, it appears that FNAC may be the only investigation needed. It provides a reliable diagnosis in patients with either obstructive or non-obstructive azoospermia. Routine estimation of FSH can be omitted from the investigative protocol in these patients. Copyright 2004 S. Karger AG, Basel Semen analysis does not consistently provide definitive and predictive value in the assessment of male fertility potential. But despite its limitations, it is a simple, inexpensive and most important screening test with which to begin the laboratory evaluation of male infertility. The proposed clinical approach is to identify recognizable causes of male infertility that are manageable or unmanageable. Routine hormonal evaluation is at best suggestive and is neither reliable nor conclusive. Hence, we feel that there is little to be gained by measuring hormonal levels in infertile people. The current teaching of male infertility recommends that men with azoospermia, normal ejaculate volume and follicule-stimulating hormone (FSH) levels may have ob- Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com 2004 S. Karger AG, Basel 0042 1138/04/0731 0023$21.00/0 Accessible online at: www.karger.com/uin Dr. Aneesh Srivastava Department of Urology and Renal Transplantation SGPGIMS, Rae Barelly Road, Lucknow-226014, UP (India) Tel. +91 522 2668004, ext. 2131, 2132, Fax +91 522 2668017 E-Mail anees@sgpgi.ac.in
structive or nonobstructive azoospermia and a diagnostic testicular biopsy is recommended in these patients [1 3]. The previously held tenet that men with greater than twice the normal FSH levels and bilateral testicular atrophy are devoid of sperm, no longer applies following retrieval of occasional sperm in such individuals which could be successfully subjected to intra-cytoplasmic sperm injection (ICSI). Further, Sobek et al. [4] have opined that the presence of high FSH level and testicular hypotrophy does not exclude any patient from undergoing testicular biopsy. Thus it seems that whereas testicular biopsy is mandatory in all cases with azoospermia, the routine use of FSH estimation is subjected to scrutiny [5, 6]. Open testicular sperm extraction is the best technique for sperm extraction. However, there are inherent disadvantages. It induces scarring which makes subsequent testicular biopsy, sperm extraction and reconstructive surgery difficult or impossible [7]. This method is also invasive and traumatic. Harrington et al. [8] reported that 29% of single open diagnostic testicular biopsies caused intratesticular hematoma formation. Percutaneous testicular core biopsy, performed with a 14 gauge needle has been proposed as an alternative method [9]. This also did not gain in popularity due to fear of trauma and hematoma. Schlegel et al. [10] reported testicular atrophy due to injury to testicular blood supply. Testicular fine-needle aspiration biopsy (FNAC) has been reported by our group as well as others as a minimally invasive and safe investigative modality in evaluation of testicular function and disease [11, 12]. We therefore preoperatively performed routine testicular FNAC in all patients with azoospermia to evaluate its impact on approach and management in this subgroup of patients. We wanted to see whether FNAC of testis alone is sufficient to diagnose testicular function and wheather FSH estimation can be safely deleted from the evaluation protocol of the azoospermic subject. We felt that testicular FNAC has the potential to divide the azoospermics into two groups: manageable and unmanageable. Material and Methods We studied 46 adult azoospermic males (24 45 years of age) with a mean age of 33.5 years who were infertile for more than 2 years following marriage. Infertile males aged less than 20 or more than 45 years were excluded from the study, as age can affect the characteristics of sperm [13]. Each person had a semen analysis done at least on 3 different occasions, separated by a 3-week interval and found to be consistently azoospermic. Azoospermic patients with prior vasectomy, failed vasovasostomy and failed epididymovasostomy were excluded from the study. There were no significant contributory findings derived from the history. There was no comorbid condition in any of the patients. Physical examination was unremarkable except for small sized testis in 6 patients and congenital absence of vas in 2 patients. Fructose was negative in 3 patients. The testicular long axis was measured bilaterally in all patients with a commercially available caliper, accurate to within 1 mm. These patients were broadly divided into two groups with a size of 4.5 cm being considered as the dividing line. Hormonal profile was ordered and data regarding FSH, LH and testosterone levels were available in all the patients. The normal ranges of FSH, LH and testosterone were 5 15 miu/ml, 5 10 miu/ ml, and 9 25 nmol/l, respectively, according to our laboratory guidelines. Patients were broadly grouped into two categories with FSH values two and a half times the upper limits of normal being used as the cut-off point. All 46 patients were then subjected to bilateral FNAC of the testes. The hospital ethical committee approved our study and informed consent was obtained from each patient. FNA was obtained under local anesthesia using a 22-gauge needle and aspirating with a 10-ml syringe. Several aspirating passes were made through a single puncture site. Three puncture sites were chosen, preferably on the anterolateral surface and away from the epididymis. The air-dried smears were stained with May-Grünwald-Giemsa method. The aspirates were considered adequate when at least 200 cells could be counted in at least 1 well spread smear. Patients diagnosed to have testicular failure on cytology were subjected to testicular biopsy, in order to compare the histopathological details with cytological features. Only nonobstructive azoospermics undergoing testicular biopsy were included for study purposes. The rest of the nonobstructive azoospermics were excluded from the study. The biopsies were read in a blinded fashion. Biopsy was done under local anesthesia and a single random biopsy was taken. Care was taken to avoid testicular vessels. All fructose positive patients with normal findings on cytology were subjected to scrotal exploration and vasoepididymal anastomoses (VEA). At the time of exploration, epididymal aspirates were examined under the microscope for the presence of spermatozoa. Initial aspirate was always obtained from the tail and subsequent ones were taken from body and head if the previous ones did not reveal any sperm. There was no particular fixed site for aspiration. Epididymal aspiration was then correlated with the earlier cytological findings. Results The cytological smears were divided into five groups. Patients whose cytological smears were interpreted as normal constituted group A. Smears showing Sertoli cell only syndrome, maturation arrest or hypospermatogenesis were included in groups B, C and D, respectively. A single patient whose cytology was inconclusive was placed in group E. Demographic details of these patients are given in table 1. There were no significant differences in cy- 24 Urol Int 2004;73:23 27 Srivastava/Raghavendran/Jain/Gupta/ Chaudhary
Table 1. Demographic profile of patients Group Interpretation Number Age years Time of diagnosis years Comorbid condition A Normal 34* 33.6 (24 45) 4.7 (3 11) 0 B Sertoli cell only sydrome 15 34.8 (34 40) 5.2 (3 8) 0 C Maturation arrest 13 31.3 (30 37) 3.3 (2.5 4) 0 D Hypospermatogenesis 13 34 (30 38) 3.7 (3 4.5) 0 E Inconclusive 11 32 4 0 Total 46 33.5 (24 45) 4.7 (2.5 11) 0 * Of the 34 patients, 29 were fructose positive and clinically normal. 3 patients were fructose negative, 2 patients had CBAVD. Figures in parentheses are ranges. Table 2. Correlation between FNAC, FSH and testicular size Group Number FSH >2.5 times of normal FSH <2.5 times of normal Testis size >4.5 cm Testis size <4.5 cm A 34 2 32 33 1 B 15 4 11 11 4 C 13 2 11 13 0 D 13 1 12 12 1 E 11 11 11 Total 46 9 37 40 6 tological findings between the right and left testicular FNAC in all 46 cases. Five patients (3 with negative fructose and 2 with bilateral congenital absence of vas deferens) and other 11 patients with non-obstructive azoospermia were subjected to testicular biopsy in order to compare the findings on FNAC. The cytological findings in all 16 cases were consistent with the findings of testicular biopsy except 1 case in group B where the final testicular biopsy specimen revealed maturation arrest. In 1 patient the aspirate was inconclusive and therefore he was also subjected to testicular biopsy which diagnosed maturation arrest. Correlation between FSH levels, FNAC and testicular size data are given in table 2. Twenty-nine (group A) patients were straightaway subjected to scrotal exploration under anesthesia. Spermatozoa could be documented from tail, body or head in all but 2 patients and these 27 patients were subjected to vasoepididymal anastomoses. The other 2 patients, in whom the spermatozoa could not be documented, were subjected to testicular biopsy and found to have normal spermatogenesis. There were no major complications encountered during the cytology procedure. Two small hematomas were seen in 2 patients which resolved in a few days. Two patients complained of vague pain at the site of aspiration which responded to oral analgesic intake in the following 24 h. All the patients were sent home after observing them for half an hour. Discussion This study differs from other studies in that there is a predominance of obstructive azoospermic patients. This does not reflect the true incidence, as in the present study only nonobstructive azoospermics undergoing testicular biopsy were included for study purposes. Patients with obstructive azoospermia underwent epididymal aspiration at the time of exploration. This was done to correlate these findings with the earlier cytological findings. We found 95.65% (44/46) agreement between FNAC and testicular biopsy. Gottschalk-Sabag et al. [14] report- FNAC May Be the Only Investigation Needed in Azoospermics Urol Int 2004;73:23 27 25
ed an 87% agreement in 51 of their 59 specimens while Odabas et al. [15]. showed agreement in 52 of 58 specimens and Person et al. [16] showed agreement in 51 of their 59 specimens. Our results may be better as compared to other series due to larger ongoing experience with time. In 1 patient, the diagnosis was inconclusive and in another the cytological diagnosis was SCO (Sertoli cell only syndrome). Both patients had maturation arrest apparent on histological sections. The histological diagnosis of SCO encompasses several patterns including Sertoli cell only, fibrosis, focal fibrosis and atrophy. It is not possible to differentiate these entities cytologically as FNAC samples only cells and hence the smears were categorized as SCO. Secondary spermatocytes may not be seen on cytological smears because of their short life span and immediate transformation to spermatids [14]. Two patients in the scrotal exploration group were not found to have spermatozoa from any part of the epididymis. This could be attributed to the technical or operatordependent problem but the findings of testicular FNAC remained consistent with subsequent testicular histology. Two patients with congenital absence of the vas deferens were aspirated and biopsied in our study. This was because both these patients had high FSH. In this group of patients, normal FSH levels indicate adequate spermatogenesis and biopsy is usually not necessary. The present study shows that even if the hormonal evaluation is abnormal, these patients do have normal spermatogenesis. There were 3 patients with fructose negativity in the study group. All the patients had normal seminal volumes. Post-ejaculatory urinalysis did not reveal any sperm. Transrectal ultrasound did not demonstrate ejaculatory duct obstruction. This is not surprising since it is well known that seminal fructose measurements have many false positives and negatives. However, given the simplicity of the test, seminal fructose measurement should be part of the screening protocol along with a wellperformed semen analysis. Classical teaching in most of the centers is that serum FSH estimation should be the initial screening test in an azoospermic male. It has been argued that if the FSH level in a patient with small testis is more than 2.5 times above normal, then the patient does not need a testicular biopsy. But we already know that in the ICSI era, it is still possible to obtain sperm from 50% of such individuals [5]. Hence, we find that obtaining FSH values in these individuals has no major diagnostic value. In the present series also, we found that FSH levels were raised in only 7 of the 11 patients with primary testicular failure. This data is proof of the unreliable nature of FSH analysis. In patients with normal-sized testis, normal FSH levels indicate adequate spermatogenesis and therefore FSH estimation has been used universally in this group. In the present study of the entire 34 group A patients, FSH levels were near normal in 32. Though FSH levels suggested adequate spermatogenesis in 32 of the 34 patients, this was proved only by the FNAC. This shows the suggestive but inconclusive nature of FSH analysis. When testicular size was taken into consideration, we found that this also could be suggestive and could never be reliable and conclusive. We feel that the only role of FSH in infertile people is the therapeutic use in patients with proven hypogonadotropic hypogonadism. These are the patients in whom McNally [17] has opined that hormonal evaluation should be performed as the history and results of physical examination indicate an endocrine problem. FSH estimation requires a well-equipped lab and an extra visit to the lab. In the scenario of developing countries, the cost of investigation may also be a considerable factor. Since FSH is at best suggestive, we feel that its other important role will be in the evaluation of infertile men in centers which lack an experienced cytopathologist. In the present series FNAC has shown good correlation in both circumstances with testicular function leading to the conclusion that FSH estimation would have been superfluous. If a testicular biopsy was done as is the usual practice to confirm diagnosis, it could lead to subsequent problems during retrieval of sperm for ICSI as reported in the literature [16]. There is an inherent bias with most of the urologists and pathologists regarding the high level of technical skill required to perform and interpret FNAC. The report by Kinia et al. [11] is very encouraging in this regard. This study showed that the cytological interpretations of smears were so simple that even the urologists can interpret them correctly. There are reports in the literature evaluating a combination of endocrine profiles and testis size or volume with an idea to obviate the need for diagnostic testicular biopsy in a majority of patients with azoospermia [18 20]. At least one of these reports date back to an era where cytology was not an established procedure [20]. In the most recently published article by Schoor et al. [18], the authors arguably convince the readers against routine use of diagnostic testicular biopsy but do not discuss anything 26 Urol Int 2004;73:23 27 Srivastava/Raghavendran/Jain/Gupta/ Chaudhary
about FNAC. A bias towards using a combination of hormonal profile and testicular size as useful parameters cannot be ruled out. It might be argued that both FNAC and FSH be omitted in the evaluation protocol, and that each patient be directly subjected to scrotal exploration. However, a patient with primary testicular failure would be unnecessarily subjected to an exploration under anesthesia and will have to bear the consequences of the same. By doing a FNAC, we can spare the patient added morbidity of anesthesia and at the same time achieve a diagnosis. Conclusions FNAC alone appears to be a quick, safe and minimally invasive modality as part of the investigative protocol following a well-performed semen analysis in an azoospermic. It provides a reliable diagnosis in patients with either obstructive or nonobstructive azoospermia. Routine estimation of FSH is at best suggestive and can be omitted from the investigative protocol in these patients in centers with large cytology experience. References 1 Jarrow J, Sigman M: Office evaluation of the subfertile male. AUA Update Ser 1999; 18: 178. 2 Sigman M, Lipshultz LI, Howards SS: Evaluation of the subfertile male; in Lipshultz LI, Howards SS (eds): Infertility in the Male, ed 3. St. Louis, Mosby, 1997, pp 173 193. 3 Coburn M, Kim ED, Wheeler TM: Testicular biopsy in male infertility evaluation; in Lipshultz LI, Howards SS (eds): Infertility in the Male, ed 3. St. Louis, Mosby, 1997, pp 219 248. 4 Sobek A, Hrbkova K, Mucha Z, Tesarova M, Priesnitz J, Zatura F, Scheinar J: Treatment of infertility in non-obstructive azoospermia using the testicular sperm extraction method. Ceska Gynekol 1998; 63: 287 291. 5 Haberman H, Seo R, Cieslak J, Niederberger C, Prins GS, Ross L: In vitro fertilization, outcomes after intracytoplasmic injection with fresh or frozen thawed testicular spermatozoa. Fertil Steril 2000; 73: 955. 6 Bourne H, Watkins W, Speirs A, Gordon-Baker HW: Pregnancies after ICSI collected by fine needle biopsy of the testis. Fertil Steril 1995; 64: 433. 7 Verma A, Basu D, Jayaram G: Testicular cytology in azoospermia. Diagn Cytopathol 1993; 9: 37 42. 8 Harrington TG, Schauer D, Gilbert BR: Percutaneous testicular biopsy: An alternative to open testicular biopsy in evaluation of the subfertile male. J Urol 1996; 156: 1647. 9 Cohen MS, Warner RS: Needle biopsy of the testis: A safe outpatient procedure. Urology 1987; 29: 279 284. 10 Schlegel PN, Su LM: Physiological consequences of testicular sperm extraction. Hum Reprod 1997; 12: 1688 1692. 11 Kinia Rammou R, Anagnostopoulou I, Tassiopoulos F, Lykourinas M: Fine needle aspiration of the testis: Correlation between cytology and histology. Acta Cytol 1999; 43: 991 998. 12 Batra VV, Khadgawat T, Agarwal A, Krishnani N, Mishra SK, Mithal A: Correlation of cell counts and indices in testicular FNAC with histology in male infertility. Acta Cytol 1999; 43: 617 623. 13 Schwartz D, Mayaux MJ, Spira A, Moscato ML, Jouannet P, Czylick F: Semen characteristics as a function of age in 833 fertile men. Fertil Steril 1983; 39: 530 535. 14 Gottschalk-Sabag S, Glick T, Weiss DB: Fine needle aspiration of testis and correlation with testicular open biopsy. Acta Cytologica 1993; 37: 67 72. 15 Odabas O, Ugras S, Aydin S, Yilmaz Y, Atilla MK: Assessment of testicular cytology by fine needle aspiration and the imprint technique: Are they reliable diagnostic modalities? Br J Urol 1997; 79: 445 448. 16 Persson PS, Christi A, Obront KO: Aspiration biopsy smears of testis in azoospermia: Cytological versus histological examination. Scand J Urol Nephrol 1971; 5: 22 26. 17 McNally MR: Male infertility endocrine causes. Postgrad Med 1987; 81: 207 213. 18 Schoor RA, Elhanbly S, Niederberger SC, Ross LS: The role of testicular biopsy in the modern management of male infertility. J Urol 2002; 167: 197 200. 19 Tounaye 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. 20 Pryor JP, Pugh RC, Cameron KM: Indications for testicular biopsy or exploration in azoospermia. Br J Urol 1978; 50: 591. FNAC May Be the Only Investigation Needed in Azoospermics Urol Int 2004;73:23 27 27
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