Testicular Touch Preparation Cytology

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Vol. 156, 1412-1414, October 1996 Printed in U.S.A.

Tm:JOURNAL OF UROLOSY Copyright 0 1996 by AMERICAN UROLOGICAL ASSOCIATION, IN(.

Urologists At Work TESTICULAR TOUCH PREP-TION EDWARD D. KIM, JOHN A. GREER, JACKI ABRAMS

CYTOLOGY AND

LARRY I. LIPsHULTz''

From the Scott Department of Urology, Baylor College of Medicine and Department of Pathology, St. Luke's Episcopal Hospital, Houston, Texas

ABSTRACT

Purpose: The applications, technique and limitations of testicular touch preparation cytology in evaluation of the infertile man are described. Materials and Methods: The technique and histological results of testicular touch preparation cytology for normal specimens and various pathological conditions of male infertility are described in detail. Results: Testicular touch preparation cytology is able to differentiate between late spermatids and mature spermatozoa, as well as to identify other spermatogenic elements. Conclusions: Touch preparation cytology h a s 2 important purposes: 1) distinguishing between maturation arrest at the late spermatid stage and normal spermatogenesis, and 2) determining whether obstruction is the cause of azoospennia. While testis biopsy is the gold standard, touch preparation cytology is a n extremely important adjunct because of its ability to define whole sperm. The urologist and pathologist are better able to make a diagnosis when routine biopsy and touch preparation cytology are performed. KEY WORDS:testis, testicular neoplasms, cytology

Testis biopsy is a simple, minimally invasive procedure with little attendant morbidity. When performed correctly, it provides the practicing urologist with important information for diagnosis and prognosis of the infertile man. Diagnostic accuracy of testis biopsy is significantly improved when touch preparation cytology is included routinely. Although this technique for obtaining cytological specimens has been cited in recent reports, to our knowledge it has not been described in detail.1.2 Even at centers that evaluate large numbers of infertile men, touch preparation cytology is not performed routinely. The inability to distinguish late spermatids from mature sperm is a significant problem with analysis of whole testis tissue prepared by routine hematoxylin and eosin staining. Because of this difficulty, it is not always possible to differentiate between maturation arrest at the late spermatid stage and normal spermatogenesis using routine histology alone. Because touch preparation cytology clearly identifies spermatids and mature sperm, the primary benefits are in distinguishing between maturation arrest at the late spermatid stage and obstruction in the azoospermic or severely oligospermic patient, especially if surgical reconstruction is planned a t biopsy. MATERIALS A N D METHODS

After a standard open testis biopsy is performed, testis touch preparation cytology is done by rapidly touching the excised testis tissue numerous times to a microscope slide (fig. 1). Gentle and proper specimen handling, involving touching rather than smearing, is important to avoid crush artifact. Immediately, before the slide has dried, cytological fixative is sprayed onto the touch preparation. Cellular arAccepted for publication March 1, 1996. * Requests for reprints: 6560 Fannin, Suite 2100, Houston, Texas 77030.

first sample

tes tissue

FIG. 1. After using standard open testis biopsy technique, testis biopsy specimen is gently grasped with jeweler's forceps and immediately touched, rather than smeared, onto multiple sites of microscope slide. Slide is immediately (within seconds) sprayed with cy-

tological fixative, before staining.

chitecture is distorted if the slide air dries before the fixative is applied. The testicular biopsy is then placed in Bouin's fixative. In the pathology laboratory (typically the frozen section room) the slide is processed with routine hematoxylin and eosin staining, and diagnosis may be available within 5 minutes. This rapid diagnosis is especially important if mi-

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FIG. 2. Normal spermatogenesis. A, open biopsy of obstructed testis demonstrates abundant cells a t all stages of spermatogenesis. Large numbers of mature sperm are probably present but tails cannot always be ascertained. B , touch preparation cytology shows numerous mature sperm with complete tails, confirming diagnosis of normal spermatogenesis. H & E, reduced from X400.

FIG. 3. Maturation arrest at primary spermatocyte stage. A, open biopsy shows abundant primary spermatocytes but late spermatids and mature sperm are absent. B , touch preparation cytology demonstrates no mature spermatozoa or spermatids. Numerous Sertoli’s cells, Leydig’s cells and primary spermatocytes in various stages of development are present. H & E, reduced from X400.

crosurgical repair of an obstructive lesion is to be performed concurrently. RESULTS

Routine hematoxylin and eosin stain and touch preparation cytology of a n obstructed testis reveal cells at all stages of spermatogenesis, with large numbers of mature sperm (fig. 2). Most maturation arrests are characterized by an abundance of primary spermatocytes, including mitotic active forms, with the notable absence of late spermatids as well as mature sperm (fig. 3).3 Maturation arrests may also present at a later stage of spermatogenesis, when spermatids are present but mature spermatozoa are absent (fig. 4). Hypospermatogenesis is characterized by the same types of cells found in the obstructed testis but with quantitatively fewer mature sperm and a decrease in all cell forms (fig. 5). Touch preparation of a testis with the Sertoli-cell-only syndrome

will show only Sertoli’s, Leydig‘s and small numbers of other somatic cells, such as leukocytes with no spermatogenic elements (fig. 6). Touch preparation cytology clearly identifies all cells within the testis. Late spermatids are easily seen as distinctly different from mature sperm by tails in the latter cases. DISCUSSION

The use of testicular touch preparation cytology greatly improves diagnostic capability when performed in conjunction with routine histological preparation of testicular tissue. Because complete, mature sperm are often difficult to identify on testis biopsy, differentiation between maturation arrest at the late spermatid stage and normal spermatogenesis can be difficult. While testis biopsy is the gold standard, touch preparation cytology is an extremely important adjunctive test in these situations because of its ability to define whole sperm.

FIG. 4. Maturation arrest a t spermatid stage. A, open biopsy reveals spermatogenic cells at all levels of develo ment but no mature sperm with tails. Densely compact nucleus of round s ermatid may be difficult to distinguish from mature sperm {ead. B , touch preparation Cytology shows no mature spermatozoa, althougz: spermatids are present. H & E, reduced from X400.

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PREPARATION CYTOLOGY

FIG. 5. Hypos rmatogenesis. A, open biopsy demonstrates marked1 fewer mature sperm and decrease in all cell forms. although cells at all levels of devecpment are present compared to obstructed testis. H E, reduced from X400. B , touch preparation Cytology shows mature sperm, which are markedly infrequent. Reduced from ~200.

FIG. 6. Sertoli-cell-only syndrome. A, open biopsy reveals only Sertoli's and Leydig's cells with no spermatogenic elements. H & E. reduced from ~ 2 0 0B. , touch preparation cytology shows only Sertoli's cells with faint nuclei. Reduced from x400.

The urologist and pathologist are better able to make a diagncsis when routine biopsy and touch preparation cytology are performed. The importance of touch preparation cytology in determining (particularly a t microsurgical reconstruction when routine hematowlin and eosin sections are not available) whether obstruction is the cause of azoospermia also cannot be overstated. An alternative to hematoxylin and eosin stain for touch preparation cytology is use of the Diff-Quik* method.2 Similar to the method presented in our study, simple and rapid identification of spermatozoa and spermatids is possible. Touch preparation cytology with the ASAP? percutaneous core biopsy system, compared to open biopsy diagnosis and open biopsy touch preparation cytology, has demonstrated excellent correlation.4 Aspiration cytology may provide a diagnostic alternative to touch preparation cytology. This technique allows the clinician to obtain testicular parenchyma percutaneously with use of local anesthesia. Among the drawbacks of this technique are that relatively few cells are obtained and, unless there is concomitant testis biopsy, there is no histological material to examine. Distinguishing between hypospermatogenesis and obstruction may be difficult, if not impossible, with aspiration cytology alone. Similarly, given its qualitative rather than quantitative nature, identifying hypospermatogenesis with touch preparation cytology is unreliable. Computer based image analysis, a technique for determining deoxyribonucleic acid cell ploidy, can be performed with a testicular aspirate or biopsy specimen. This technique is ideally suited for use with small numbers of cells, and the slide smear can be performed at the touch preparation. Recent investigation suggested that image analysis using testicular aspirates is an objective qualitative and quantitative

* Baxter, Utrecht, The Netherlands.

I'Travenol Laboratories, Deerfield, Illinois.

method for evaluation of spermatogenesis." A distinct possibility for the future is performance of aspiration cytology for touch preparation and image analysis in lieu of open biopsy in select patients. CONCLUSIONS

The testis touch preparation is a rapid and simple procedure that can be performed a t routine testis biopsy, and should be considered an integral part of all testis biopsies. The principal benefits are in distinguishing between maturation arrest at the late spermatid stage and obstruction in the azoospermic or severely oligospermic patient, particularly a t surgical reconstruction. The improvement in diagnostic accuracy afforded by this technique makes it n worthwhile procedure in the evaluation of the infertile man. REFERENCES

1. Coburn, M. and Wheeler, T. M.: Testicular biopsy in male infertility evaluation. In: Infertility in the Male, 2nd ed. Edited by L. I. Lipshultz and S. S. Howards. St. Louis: Mosby Year Book, chapt. 11, pp. 223-253, 1991. 2. Belker, A.M., Sherins, R. J. and Dennison-Lagos. L.: Improved, simple and rapid staining method for intraoperative examination of testicular biopsy touch imprints. Fertil. Steril., suppl., S128,abstract P-074,1995. 3. Rudy, F.: Male infertility. In: Uropathology. Edited by G. S. Hill. New York: Churchill Livingstone, chapt. 25, pp. 1002-1033, 1989. 4. Kessaris, D. N.,Wasserman, P.and Mellinger, B. C.: Histopathological and cytopathological correlations of percutaneous testis biopsy and open testis biopsy in infertile men. J. Urol., 153: 1151,1995. 5. Gottschalk-Sabag, S.,Weiss, D. B. and Sherman, Y.: Assessment of spermatogenic process by deoxyribonucleic acid image analysis. Fertil. Steril., 64:403,1995.