Functional and cytologic features of the contralateral testis in cryptorchidism

Transcription

1 FERTILITY AND Copyright Ci') 1996 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Functional and cytologic features of the contralateral testis in cryptorchidism Carlo Foresta, M.D.*t Alberto Ferlin, M.D. * Andrea Garolla, M.D. * Claudio Milani, M.D.:\: Gaetano Oliva, M.D.:\: Marco Rossato, M.D. * University of Padova, Padova, Italy Objective: To characterize the testicular cytologic pictures in cryptorchid and contralateral testis related to seminal pattern. Design: Controlled clinical study. Setting: Andrological and urologic academic setting. Patients: One hundred nine patients orchidopexied because of unilateral cryptorchidism compared with 35 normospermic subjects. Interventions: Bilateral testicular fine-needle aspiration cytology. Main Outcome Measures: Seminal parameters; testicular cytologic features; plasma levels of FSH, LH, and T; and ultrasound testicular examination. Results: The cytologic analysis revealed an important quantitative and qualitative impairment of spermatogenic line in all the excryptorchid testes. These alterations are present in the contralateral testes only when azoospermia or severe oligozoospermia were observed, whereas in moderate oligozoospermic subjects a normal tubular status was evident. A compensatory activity in normally descended testis of normozoospermic patients was present. Conclusions: In unilateral cryptorchidism, a frequent testicular damage in the orchidopexied and in the contralateral descended testis suggests that this condition is the end point of different pathological conditions, including testicular intrinsic (congenital) and extrinsic (anatomical) causes. Fine-needle aspiration cytology of both testes represents a tool in the assessment of the tubular status in excryptorchid subjects. Fertil Steril 1996;66:624-9 Key Words: Cytology, cryptorchidism, testis, contralateral, infertility, fine needle aspiration Impaired fertility is a frequent sequelae of bilateral and unilateral cryptorchidism. It has been estimated that as many as 50% of unilateral and 75% of bilateral cases are associated with fertility problems (1, 2). Impairment of fertility in bilateral cryptorchidism is related to a well-known parenchymal damage involving both testes. In unilateral cryptorchidism, the evidence of severe impaired sperm production up to azoospermia suggests that, in addition to the cryptorchid testis, the normally descended testis may be altered too. An alteration of the contralateral testis in unilateral cryptorchidism has been ob- Received January 24, 1996; revised and accepted May 22, * Patologia Medica III. t Reprint Requests: Carlo Foresta, M.D., Patologia Medica III, Via Ospedale 105, 35128, Padova, Italy (FAX: ). :j: Istituto di Urologia. served by several authors evaluating histologic specimens from children undergoing orchidopexy at different ages (3). Nevertheless, these studies reached controversial conclusions because it has been reported a compensatory testicular hypertrophy (4, 5) as well as a progressive depopulation of germ cell line (6,7). Furthermore, in adult excryptorchid subjects, the tubular function of the contralateral testis is not well defined. The successful development of testicular fine-needle aspiration cytology in the assessment of the state of the seminiferous epithelium (8-10) prompted us to perform this procedure in a group of unilateral excryptorchid subjects to characterize the testicular tubular status related to the seminal pattern. MATERIALS AND METHODS Our study was approved by the Hospital Ethical Committee, and informed consent was obtained from 624 Foresta et al. Testicular cytology in cryptorchidism Fertility and Sterility

2 Table 1 Age of Orchidopexy and Hormonal Values of the 109 Unilateral Excryptorchid Patients Classified for Sperm Concentration Compared With Controls* Azoospermia Severe oligospermia ("do X 10 6 /ml) Moderate oligospermia (10 to 20 x 10 6 /ml) Normospermia (>20 X 10 6 /ml) Controls Patientst Age of orchidopexy (y) FSH (miu/ml) LH (miu/ml) T (nmolll) 15 (13.7) 11.9 ± ± 3.H 5.0 ± ± (35.8) 10.7 ± ± ± ± (12.8) 9.8 ± ± ± ± (37.7) 7.2 ± ± ± ± ± ± ± 3.1 * Values are means ± SD. Conversion factors to SI units are as follows: FSH and LH, 1.00; T, t Values in parentheses are percentages. :j: P < versus controls. P < 0.01 versus controls. each patient. We have evaluated 109 subjects who underwent orchidopexy because of unilateral cryptorchidism, aged 18 to 28 years (23.2 ± 3.5 years; mean ± SD). All patients were studied with a comprehensive general investigation for exclusion of varicocele, antisperm autoantibodies, seminal tract infections, postmumps orchitis, testicular trauma or torsion, drugs assumption, endocrinopathies, and systemic diseases. All subjects were unmarried and they did not complain of a history of infertility. The age of orchidopexy is reported in Table 1; 22 of these subjects underwent surgical orchiectomy because of testicular atrophy. As controls, we studied 35 agematched subjects (24.3 ± 4.0 years) affected by idiopathic or autoimmune infertility, with normally descended testes and normal sperm count. In all patients physical examination, seminal analysis, plasma levels determination of FSH, LH, and T, and bilateral testicular ultrasound examination for testicular volume (by mean of ellipsoid volume formula) were performed. Semen samples were collected on two different occasions, separated by a 3-week interval after 3 days of abstinence. Follicle-stimulating hormone and LH plasma levels were measured in each subject by RIA using 125I_Ia_ beled LH and FSH and double monoclonal antibody (Ares-Serono, Milan, Italy). Interassay and intraassay variation coefficients for LH were 2.8 and 3.7, whereas for FSH they were 2.6 and 3.6, respectively. Plasma levels of T were determined using the double antibody RIA; intra-assay and interassay coefficients of variation were 7.8 and 7.0, respectively. Furthermore, we performed percutaneous fineneedle aspiration cytology on surgically and spontaneously descended testis in each patient. Mter the aspiration, each subject was investigated for complications, such as bleeding, hematocele, hydrocele, or infection. Rising of antisperm autoimmunity was excluded previously (8). Fine-needle aspiration cytology procedure was described in detail in our previous studies (8, 9). Briefly, using 23-gauge (0.6 mm) needles and 20-mL syringes, the testicular cell material was aspirated and placed on two or more glasses for each testis, allowed to air dry for 24 hours, and stained with May Grunwald-Giemsa. The cytologic evaluation was performed using a light microscope (Wild Leitz, Wetzlar, Germany) at 125x, 400x, and 1,250x magnifications, and spermatogenic status was analyzed counting :2:200 spermatogenic cells per smear. The different germ cell types were classified as spermatogonia, primary and secondary spermatocytes, round and elongated spermatids (corresponding to spermatids a-b and c-d, respectively), and spermatozoa, according to their staining and morphological aspects, such as cell diameter, chromatin pattern, and cytoplasm size as described previously (9). The number of each germ cell type was expressed as relative percentages. Sertoli cells were expressed as Sertoli Index (SEI = number of Sertoli cells/number of spermatogenic cells per 100), which has been proven previously to be a reliable index of the tubular cell potential (8, 9). The percentage of mature spermatozoa was expressed as Spermatic Index (SI), which indicates the number of spermatozoa per 100 spermatogenic cells (9). Studies regarding the reproducibility of cytologic analysis have been performed previously and reported very low coefficients ofvariation between differential counts, as well as tight agreement with the histologic findings (9). The results are given as means ± SD. Statistical significance was verified with Student's t-test. Probability (P) values < 0.05 were regarded as statistically significant. RESULTS Depending upon sperm concentration, the 109 cryptorchid patients were classified as azoospermic (13.7%), severe oligospermic (sperm count < 10 X lo B /ml) (35.8%), moderate oligospermic (sperm Foresta et a!. Testicular cytology in cryptorchidism 625

3 Figure 1 Cytologic picture in a case ofnormozoospermic subject (control): all testicular cell subtypes are seen. (May-GrunwaldGiemsa stain; magnification, xl,250). count between 10 and 20 X 106/mL) (12.8%), and normospermic (sperm count> 20 X 106/mL) (37.7%). Table 1 reports the age of orchidopexy and hormonal values observed in each group. The age of orchidopexy suggests that precocious surgical intervention results in better seminal parameters than those of subjects that underwent orchidopexy at an older age; however, the correlation between age at surgery and sperm concentration was not statistically significant. Follicle-stimulating hormone plasma levels were significantly higher in azoospermic and severe oligospermic subjects compared with controls. On the contrary, no significant differences were observed in LH and T plasma levels in each group. No complications were reported after percutaneous aspiration of the testis, as reported previously (9). Figure 1 shows a typical picture observed in a normospermic subject. Figure 2 reports the histogram of quantitative analysis of spermatogenesis, showing a constant pattern characterized by increasing percentages of cells from spermatogonia to spermatids, according to the normal maturative progression of spermatogenesis. The Sertoli:spermatogenic cells ratio, defined as the Sertoli Index, constitutes an attempt to simplify the understanding of the cytologic results. Because the number of Sertoli cells is constant per unit of tubular length, an increase of Sertoli Index reveals the existence of tubular germ cells depopulation. The ratio of mature spermatozoa to total spermatogenic cells (Spermatic Index) point out the final maturation step and the spermiogenic process. Cytologic analysis was performed in the excryptorchid and in the normally descended testis sepa- 626 Foresta et a1. Testicular cytology in cryptorchidism rately, distinguishing subjects who underwent orchidopexy (87 patients) from patients who underwent orchiectomy (22 patients). Table 2 reports the cytologic and seminal parameters in these two groups of patients. In orchidopexied subjects, the volume of the cryptorchid testis was reduced regardless of the different seminal pattern (P between 0.01 and 0.001), whereas the volume of the contralateral testis resulted reduced only in those cases showing azoospermia or severe oligozoospermia (P between 0.05 and 0.01). The cytologic picture of the cryptorchid testis showed a profound quantitative reduction of the spermatogenic line as revealed by an increase ofsertoli Index and a reduction of Spermatic Index. In azoospermic and severe oligospermic subjects, the Sertoli Index was increased in both testes (P < versus controls), showing in some cases a picture compatible with Sertoli cell-only syndrome. In moderate oligospermic subjects, the cytologic analysis of the contralateral testis did not show a germ cell depopulation. On the contrary, the descended testis showed a Sertoli Index lower than controls (P < 0.05) in normospermic subjects, demonstrating an increase of the percentage of spermatogenic cells. In this case, the normal value of the Spermatic Index demonstrated a normal spermatogenic process. In orchiectomized patients, the alterations of seminal pattern (12.7% azoospermic, 36.7% severe oligospermic, 12.7% moderate oligospermic, and 37.9% 4 gonia Spermatids cytes (Sc-Sd) Figure 2 Cell types as observed in cytologic smears of normozoospermic subjects: increasing percentages from immature to mature cells agree with the maturative progression of spermatogenesis. The low percentage of secondary spermatocytes is explained with the shorter life span of these cells (24 hours) compared with that of primary spermatocytes (2)2 weeks). Fertility and Sterility

4 -' Table 2 Testicular Volume and Cytologic Indexes Related to Seminal Pattern of the 109 Unilateral Excryptorchid Patients Distinguished in Orchidopexied and Orchiectomized Compared With Controls Orchidopexy (n = 87) Orchiectomy (n = 22) Undescended testis Descended testis Descended testis Volume SEl Sl Volume SEl Sl Volume SEl Sl Azoospermia (n = 11; 12.7%) Severe oligospermia t (n = 32, 36.7%) Moderate oligospermiat ml 9.2 :!: 2.7* 9.4 :!: 3.6* ml 1,141 :!: :!: :!: :!: :!: :!: 2.8~ ml 771 :!: :!: 1O :!: :!: :!: 10.2:1: 207 :!: :!: :!: :!: :!: (n = 11, 12.7%) 9.9 :!: :!: :!: :!: :!: :!: 23.5~ 18.9:!: :!: :!: 3.2 Normospermiat (n = 33, 37.9%) 1O.2:!: :!: :!: :!: :!: 8.1~ 49.4:!: :!: 3.8~ 19.1:!: 14.2~ 40.3:!: 7.7 Controls (n = 35) 14.5:!: :!: :!: :!: :!: :!: :!: :!: :!: 10.3 * Sertoli index, SErtoli cells/spermatogenic cells X 100; spermatic index, mature spermatozoa/spermatogenic cells X 100. t Severe oligospermia, s 10 X 10 6 spermatozoa/ml; moderate oligospermia, 10 to 20 X 10 6 spermatozoa/ml; normospermia, >20 X 10 6 spermatozoa/ml. * P < versus controls. P < versus controls. lip < 0.01 versus controls. ~ P < 0.05 versus controls. normospermic) were similar to those observed in orchidopexied subjects (18.2% azoospermic, 31.6% severe oligospermic, 13.8% moderate oligospermic, and 36.4% normospermic). In this group, the cytologic analysis of azoospermic and severe oligospermic subjects evidenced an important hypospermatogenesis (elevated Sertoli Index, P < versus controls). The testicular cytologic picture was normal in moderate oligospermic patients and it was evident by an increase of the testicular volume (P < 0.05) and a reduction of the Sertoli Index values versus control (P < 0.05) normospermic subjects, suggesting the presence of testicular hypertrophy related to an increase of the spermatogenic component. DISCUSSION The most important sequelae of cryptorchidism is the profound effect on fertility (1). It is well known that the cryptorchid testis shows an impairment of tubular function and structure as early as 2 years after birth (3, 11) and this functional and histologic damage becomes more evident with increasing patient's age. In these subjects, a progressive decrease in tubular diameter, peritubular fibrosis, and reduction of spermatogonia were identified (12). Cryptorchid testes of adults often are reduced in size and show typical morphological pictures such as tubular atrophy (due to the reduction of germinal epithelium), immaturity of Sertoli cells, and peritubular fibrosis (7). The normally descended testis of unilateral cryptorchid children may show spermatogonial alterations and tubular disfunction similar to those found in the cryptorchid testis (6). These findings suggest that cryptorchidism may represent the end point of different pathological conditions, including intrinsic (gonadal) and extrinsic (extragonadal) causes, and that the contralateral descended testis in unilateral cryptorchidism also may be dysgenetic. This hypothesis is supported further by evidence that unilateral cryptorchidism may be associated with severe impairment of sperm production up to azoospermia (13) and that testicular cancer may originate from the contralateral, not retained testis (14). At yet little is known about morphological structure of the normally descended testis in adult unilateral excryptorchid subjects and the few data available report alterations in the number and quality of spermatogonia in some cases. Previous studies on structure of cryptorchid testes have been performed by surgical biopsy (3, 15, 16). Because this technique represents an invasive procedure with potential complications (17,18), it has been recommended only in the early diagnosis of malignant tumors (carcinoma in situ) (19), even if this procedure could be important for the diagnosis and treatment of infertility. Recently, the successful development of different needle bioptic and cytologic procedures (20, 21) has provided alternative tools to open surgical biopsy in the assessment of the status of seminiferous epithelium. These methods are rapid, not very traumatic, and can be performed without anesthesia (20). Our previous studies on testicular fine-needle aspiration cytology (8, 9) prompted us to evaluate the spermatogenetic process in a large group of unilateral excryptorchid subjects. The cytologic analysis performed in both testes Foresta et al. Testicular cytology in cryptorchidism 627

5 allowed us to determine the tubular status related to sperm concentration. Cytologic analysis in the excryptorchid testis frequently showed an important quantitative reduction of the spermatogenic line as revealed by the increase of the Sertoli Index. This cytologic feature represents the histologic picture of Sertoli cell-only syndrome or severe hypospermatogenesis. According to these findings, the gonadal volume always is reduced significantly. Therefore, in these cases, seminal sperm concentration seems to be related overall to tubular function of the contralateral testis. In fact, in azoospermic and in severe oligozoospermic subjects, the cytologic picture of contralateral testis shows alterations similar to those observed in the excryptorchid testis. In these subjects, the testicular volume was reduced in both testes and FSH plasma levels were increased. In the contralateral testis of moderate oligozoospermic and normozoospermic subjects, the cytologic analysis revealed the presence of a normal spermatogenic line, whereas, in normospermic subjects, the ratio between Sertoli and spermatogenic cells is even lower than normal, suggesting the presence of a compensatory activity of the tubular function. The pathogenesis of the tubular damage observed in the contralateral testis of some patients is still unknown. These abnormalities do not depend on the negative influence of the altered cryptorchid testis, because similar seminal and cytologic characteristics were observed also in the contralateral testis of orchiectomized patients. Furthermore, the testicular cytologic alterations were not related to the age of orchidopexy. No statistical difference was observed between the age of orchidopexy in subjects showing normal spermatogenesis and those showing tubular alterations. However, the findings ofthis study do not show any relationship between the age of orchidopexy and the degree of tubular damage because only few patients underwent orchidopexy when younger than 5 years. Therefore, it is possible that, in presence of bilateral tubular damage, the cryptorchidism might be caused by genetic or congenital intrinsic malformation of the testis, whereas, when tubular function in the contralateral testis is normal, the pathogenesis of cryptorchidism may be related to anatomical defects. This hypothesis may justify the hypertrophy of descended testis that is observed also in the contralateral testis of patients affected by congenital monorchidism (22). Despite that the pathogenesis of cryptorchidism may include different mechanisms (mechanical, endocrinologic, dysgenetic), the persistence of the testis in the abdomen can cause or worsen the tubular damage indeed. Therefore, an early orchidopexy appears to be helpful and advisable in all cryptorchid patients. In conclusion, the frequent presence of testicular damage in the orchidopexied testes and of concomitant tubular alterations in the contralateral descended testis suggests that cryptorchidism is the end point of different pathological conditions. In the presence of a bilateral testicular damage, the pathogenesis of unilateral cryptorchidism may be related to testicular intrinsic causes (congenital alterations). When contralateral testis exhibits a normal functional status, cryptorchidism may depend on extrinsic causes (anatomical defects). The presence of contralateral testis hypertrophy strengthens this hypothesis. In all cases, seminal pattern seems related to the status of the normally descended testis. Fine-needle aspiration cytology of both testes could represent a tool for the clinician in the assessment of the tubular status in cryptorchid subjects, above all when they are azoospermic. Cytologic analysis frequently shows the presence of some mature spermatozoa in these patients. Therefore, in these subjects, transcutaneous testicular fine-needle aspiration could allow the retrieval of few mature spermatozoa to be used for recently introduced assisted reproductive techniques, namely intracytoplasmic sperm injection. REFERENCES 1. Kogan SJ. Fertility in cryptorchidism: an overview in Eur J Pediatr 1987; 146:S Chilvers C, Dudley NE, Gough MB, Jackson NB, Pike NC. Undescended testis: the effect of treatment on subsequent risk of subfertility and malignancy. J Pediatr Surg 1986;21: McAleer 1M, Packer MG, Kaplan GW, Scherz HC, Krous HF, Willman GF. Fertility index analysis in cryptorchidism. J Urol 1995; 153: Kirby RS, Chapple CR, Ward SP, Williams C. Is the scrotal testis normal in unilateral cryptorchidism? Br J Urol 1985;57: Farrington GH. Histological observations in cryptorchidism. The congenital germinal cell deficiency of the undescended testis. J Pediatr Surg 1969;4: Codesal J, Paniagua R, Queizan A, Santamaria L, Nistal M. Cytophotometric DNA quantification in human spermatogonia of cryptorchid testes. J Urol 1993; 149: Tarter TH, Kogan SJ. Contralateral testicular disease after unilateral testicular injury: current concepts. Semin Urol 1988;6: Foresta C, Varotto A, Scandellari C. Assessment of testicular cytology by fine needle aspiration as a diagnostic parameter in the evaluation of the azoospermic subject. Fertil Steril 1992;57: Foresta C, Varotto A. Assessment of testicular cytology by fine needle aspiration as a diagnostic parameter in the evaluation ofthe oligospermic subject. Fertil Steril1992;58: Foresta C, Ferlin A, Bettella A, Rossato M, Varotto A. Diag- 628 Foresta et al. Testicular cytology in cryptorchidism Fertility and Sterility

6 nostic and clinical features in azoospermia. Clin Endocrinol (Ox ) 1995;43: Cendron M, Keating MA, Huff DS, Koop CE, McSnayder H, Duckett JW. Cryptorchidism, orchidopexy and infertility: a critical long-term retrospective analysis. J Urol 1989; 142: Kogan SJ, Tennebaum S, Gill B, Reda E, Levitt SB. Efficacy of orchidopexy by patient age 1 year for cryptorchidism. J Urol1990;144: Fallon B, Kennedy TJ. Long-term follow-up of fertility in cryptorchid patients. Urol1985;25: Giwercman A, van der Maase H, Skakkebaek NE. Epidemiological and clinical aspects of carcinoma in situ of the testis. Eur Urol 1993;23: Hadziselimovic F, Herzog B, Buser M. Development of cryptorchid testes. Eur J Pediatr 1987;146 Supp12:S Hedinger CEo Histopathology of undescended testes. Eur J Pediatr 1982; 139: Hendricks FB, Lambird PA, Murphy GP. Percutaneous needle biopsy of the testis. Fertil Steril 1969;20: Rajfer J, Binder S. Use of biopsy gun for transcutaneous testicular biopsies. J Urol 1989; 142: Bruun E, Frimodt-Moller C, Giwercman A, Lenz S, Skakkebaek NE. Testicular biopsy as an outpatient procedure in screening for carcinoma in situ: complications and the patient's acceptance. Int J Androl 1987; 10: Persson PS, Ahren C, Obrant KO. Aspiration biopsy smear of testis in azoospermia: cytological versus histological examination. Scand J Urol Nephrol 1971;5: Rajwanshi A, Indudhara R, Goswami AK, Rodhika S, Das A, Sharma SK, et al. Fine needle aspiration cytology in azoospermic males. Diagn Cytopathol 1991;7: HuffDS, Snyder HM, Hadziselimovic F, Blyth B, Duckett JW. An absent testis is associated with contralateral testicular hypertrophy. J Urol1992;148: Foresta et ai. Testicular cytology in cryptorchidism 629