FERTILITY AND STERILITY Copyright e 1987 The American Fertility Society Printed in U.S.A. Aspiration flow cytometry of the testes in the evaluation of spermatogenesis in the infertile male*t David G. Kaufman, M.D.:j: Harris M. Nagler, M.D. Columbia-Presbyterian Medical Center, College of Physicians and Surgeons, New York, New York Deoxyribonucleic acid (DNA) flow cytometry oftesticular tissue has been demonstrated to be a quantitative means of assessing spermatogenesis. This study evaluates testes aspirates and DNA flow cytometry in the evaluation of the infertile male. Testicular tissue obtained from 12 men who underwent bilateral orchiectomy for prostate cancer (group 1) were examined by both flow cytometry and standard histologic technique to assess the correlation between these two modalities. Thirteen men evaluated for infertility (group 2) and requiring histologic evaluation of spermatogenesis underwent both open biopsy and fine needle aspiration of their testes. Histology was independently examined and grouped according to standard nomenclature. Flow cytometric analysis revealed characteristic patterns in the relative numbers of haploid (1C), diploid (2C), and tetraploid (4C) cells. These patterns correlated reproducibly with the histologic diagnoses. DNA flow cytometry of testicular aspirates provides a rapid and reliable quantitative means of assessing spermatogenesis. Fertil Steril48:287, 1987 Testes biopsy has long been used to assess spermatogenesis in infertile men. 1 It is indicated in the workup of the azoospermic (and some oligospermic) male (less than 10,000,000 sperm/ml) with normal endocrinologic parameters. 2 Testicular biopsy can differentiate between testicular and post-testicular (obstructive) causes of infertility. When post-testicular azoospermia or severe oligospermia is demonstrated, surgical correction may be indicated. Received February 2, 1987; revised and accepted April 15, 1987. *Received First Prize for Research at the F.C. Valentine Urology Residents Essay Meeting, New York Academy of Medicine, April 9, 1986. t Presented at the forty-second annual meeting of The American Fertility Society and the eighteenth annual meeting of the Canadian Fertility and Andrology Society, September 27 to October 2, 1986, Toronto, Ontario, Canada. :j: Department of Urology, Squire Urological Clinic, Columbia-Presbyterian Medical Center, New York, New York. Reprint requests: Harris M. Nagler, M.D., Associate Professor of Urology, College of Physicians and Surgeons, 630 168th Street, New York, New York. A testicular biopsy is most often performed using the surgical "window" approach under local anesthesia in an outpatient operating facility. 3 A limitation of this diagnostic technique resides in its reliance on conventional pathologic interpretation of spermatogenesis. Histologic reporting is limited by subjective analysis and is therefore often observerdependent. Recently, needle biopsy of testicular tissue has been advocated for the evaluation of male infertility.4 5 This technique is reported to offer the advantages of: (1) ease of procurement with minimal patient discomfort, (2) negligible operative risk, and (3) reduced cost. Its limitations include a small sampling volume with the potential for distortion of tubular histology. Perhaps even more importantly, this technique does not obviate the need for standard histologic evaluation. Attempts have been made to circumvent these limitations through the use of DNA flow cytometry. Flow cytometry rapidly and objectively distinguishes and quantitates cell populations on the basis of differences in DNA content. Testicular tissue is particularly well suited Kaufman and Nagler Testes aspirates 287
for evaluation by this technique. In the adult testicle with normal spermatogenesis, there appears to be an optimal ratio between populations of haploid (lc), diploid (2C), and tetraploid (4C) cells. Alterations in these ploidy relationships may indicate disordered spermatogenesis. Several studies have established the ability of flow cytometry to distinguish between normal and abnormal testicular tissue. 6-8 However, no one study has yet correlated the results of flow cytometry of fine needle aspirates with the results of flow cytometry oftissue obtained via standard open testicular biopsy. Furthermore, no study has yet correlated these results with standard histologic assessment. In order to preferentially use testes aspiration flow cytometry, these correlations must be established. In addition, it is important to obtain histologic confirmation of the diagnoses made using flow cytometry of needle aspirates before employing the latter as the recommended means of testes biopsy. MATERIALS AND METHODS Twenty-five patients undergoing either bilateral orchiectomy for treatment of stage D carcinoma of the prostate (group 1, n = 12) or testes biopsy for workup of infertility (group 2, n = 13) were entered into this study. Patients in group 1 ranged in age between 55 and 92 years (mean, 76 years); all had fathered children. Bilateral orchiectomy was performed via a scrotal approach. Immediately following removal of the testicles, a fine needle aspirate was obtained of each testes using a 20-gauge needle and a 10-ml syringe. The aspirated material was rinsed from the syringe with propidium iodide (PI), a DNA flourochrome, into a collection tube. The tunica albuginea of each testes then was incised and two small portions of extruded tubules were atraumatically excised using a "no-touch" technique. Seminiferous tubules were gently lifted away from the testicle, before being cut, to avoid disruption of tubular architecture. One biopsy was placed in Bouin's solution for histologic interpretation and the second biopsy was placed in Hank's solution for flow cytometric assessment. The 13 patients (group 2) being evaluated for infertility underwent standard hormonal assessment (luteinizing hormone [LH], follicle-stimulating hormone [FSH], and testosterone [T]) as well as a minimum of two semen analyses. Patients were between 19 and 36 years old (mean, 29) and all had at least 1 year of unprotected sexual intercourse. Six patients had clinical varicoceles; three were determined to have obstructive azoospermia on the basis of history or physical examination; two patients had a history of cryptorchidism; and two had no significant urologic history. Approval for this study was granted by the Institutional Review Board and informed consent was obtained from each patient. Prior to testes biopsy, a fine needle aspiration of each testicle was performed under direct vision. Open testes biopsy was obtained and prepared for flow cytometric assessment and routine histologic evaluation, as described for group 1. Local anesthetic was employed for all biopsies, except when carried out at the time of varicocelectomy, when epidural anesthesia was used (n = 6). Flow Cytometry Specimens for flow cytometry were placed into single-cell suspension by means of mechanical dispersion. These cells were then suspended in PI (50 ~g/ml of PI in a 10-2 M Tris buffer, ph 7.0, with 5 mm MgCl 2 and 1 mg/ml sodium azide), which specifically stains nuclear DNA. To obtain pure DNA, samples are treated with ribonuclease (RNAse) to remove RNA contaminants (10 ~l of 6 mg/ml RNAase is added to 1 ml of cell suspension). The sample is incubated at 37 C for 2 hours, filtered through a 74-~m nylon mesh, and submitted for flow cytometric analysis, which is performed on a Coulter Epics V flow cytometer (Coulter Corporation, Hialeah, FL) equipped with an argon laser. The PI-stained cell suspension is passed single file through the beam of the argon laser. Light excitation of the stained DNA elicits a flourescent emmission. The signal is converted to an electrical impulse that is processed and recorded as a DNA histogram. Each peak on the histogram represents a cell population with a specific DNA content. Computerized analysis allows for the rapid determination of DNA content in large numbers of cells. Curve analysis was performed with the curve integration programs provided by the Coulter Multiparameter Data Acquisition and Display Software, with the analyzer determining the upper and lower limits of each ploidy compartment (histogram peak). A reference sample of known diploid cells (PI-stained lymphocytes) allows for the appropriate designation of the various ploidy peaks generated. 288 Kaufman and Nagler Testes aspirates Fertility and Sterility
Group 1 RESULTS Histologic assessment of the testes biopsies in this group revealed normal spermatogenesis in six patients, reduced spermatogenesis in two, and severe atrophy with minimal or no active spermatogenesis in four patients. All specimens in this group were noted to contain occassional sclerosed tubules and focal fibrosis. DNA histograms were generated from open and aspiration testicular biopsy specimens. The relative number of cells in each ploidy compartment was determined by computer analysis, as described previously. Histogram patterns were generated by ranking each ploidy compartment in decreasing order (from greatest percentage of cells to least). Six open biopsy flow cytometry specimens shared a ploidy relationship of 1C > 2C > 4C cell populations. Histologic evaluation of each of these specimens revealed a diagnosis of normal spermatogenesis. In four open biopsy specimens, ranking yielded a relationship in which 2C > 4C > 1C. Each of the specimens with this ploidy relationship demonstrated minimal or no spermatogenesis with atrophy, using histologic techniques. In two patients, a 2C ;;::: 1C > 4C ploidy pattern was noted. Histologic assessment of these biopsies revealed reduced spermatogenesis. When the flow histograms of aspirated testicular tissue were compared with those generated from t Haploid!! -G) 0 0 Diploid -;g 0 1c 2c 4c [ 1C>2C >4C] Figure 1 Example of DNA histogram of testes aspirate demonstrating a ploidy relationship of lc > 2C > 4C, consistent with normal spermatogenesis as assessed by standard histologic techniques. -0 '$. Haploid 1c Diploid 2c 4c [2C~1C >4C] Figure 2 Example of DNA histogram of testes aspirate demonstrating a ploidy relationship of 2C ~ lc > 4C, consistent with partial maturation arrest or hypospermatogenesis on routine histology. the open biopsy specimens, an excellent correlation was observed: r < 0.89 for haploid cells, r < 0.96 for diploid, and r < 0.89 for tetraploid cells; P < 0.01). Group 2 Histologic assessment of the testicular biopsies in the infertile group demonstrated normal spermatogenesis in three patients, partial maturation arrest or hypospermatogenesis in six patients, Sertoli-cell-only syndrome in one patient, and complete maturation arrest in three patients. Flow cytometric analysis of the open testes biopsy specimens obtained from these patients were carried out as described previously. A ploidy relationship of 1C > 2C > 4C was seen in the DNA histograms of each of the three patients with normal spermatogenesis on histologic evaluation. A ploidy relationship of 2C;;::: 1C > 4C was observed in each of the six patients with partial maturation arrest or hypospermatogenesis. The three patients with a histologic diagnosis of complete maturation arrest exhibited the ploidy relationship of 2C > 4C > 1C. A subpopulation of this latter group had no cells in the haploid or tetraploid compartment (i.e., 2C = 100%), corresponding to the single patient with Sertoli-cell-only syndrome. The histograms generated from the testicular aspirates maintained the identical ploidy relationships (Figs. 1, 2, and 3) discussed previously. Furthermore, the correlation of relative numbers of cells within each ploidy compartment or histogram peak between the aspiration and open biopsy speci- Kaufman and Nagler Testes aspirates 289
mens were found to be excellent (haploid, r = 0.95, P < 0.05; diploid, r = 0.99, P < 0.01; and tetraploid, r = 0.86, not significant). DISCUSSION To show that flow cytometric evaluation of testicular aspirates can replace standard histologic evaluation of open testicular biopsy, two conditions must be met. First, it must be shown that the DNA histograms obtained from aspiration biopsy accurately correlate with those obtained from DNA histograms of open testes biopsy. Second, flow cytometric analysis must be shown to provide equivalent clinical information as that obtained from histologic assessment so that clinical decisions regarding management can be made. Only then can one safely suggest the use of flow cytometry of testicular aspirates for the assessment of spermatogenesis in the infertile male patient. Our data demonstrate an excellent correlation between the flow cytometric analysis of testicular tissue obtained by standard open biopsy and as piration techniques. Furthermore, there are constant flow cytometric relationships characteristic of specific histologic diagnoses (Fig. 4). In the presence of normal spermatogenesis, the haploid compartment contained the majority of cells, followed by the diploid, and then tetraploid compartments (1C > 2C > 4C). In patients with complete maturation arrest on histologic assessment of open testes biopsy, DNA histograms demonstrated predominantly di- Diploid 1c 2c 4c (2C >4C >1C) Figure 3 Example of DNA histogram of testes aspirate demonstrating a ploidy relationship of 2C > 4C > lc, consistent with complete maturation arrest on routine histology. 100 90 10 1C 2C 4C Normal Spermatogenesis Hypospermatogenesisf Partial Maturation Arrest tc 2C4C Complete Maturation Arrest 1C2C 4C Sertoli Cell Only Syndrome 1C 2C 4C Figure 4 Bar graph demonstrating the four major ploidy relationships observed with the respective histologic diagnoses. ploid, followed by tetraploid, and then haploid cells (2C > 4C > 1C). Sertoli-cell-only syndrome demonstrated the exclusive presence of diploid cells (2C = 100% ). Partial maturation arrest and hypospermatogenesis shared the ploidy relationship of 2C ~ 1C > 4C. With the recognition and use of the ploidy relationships obtained from flow cytometric analysis of testicular aspirates, appropriate therapeutic decisions can be made. The use of this technique appears to provide the clinician with a convenient and effective means of assessing spermatogensis in the infertile male. Clinicians without direct access to a flow cytometer can forward their aspiration specimens to tertiary care centers with this tool. DNA histograms can provide a rapid and objective assessment of spermatogensis. Though DNA flow cytometric analysis of testes aspirates does not provide information regarding tubular or intersitial morphometry, its quanitification of spermatogenesis appears to permit appropriate management decisions in the treatment of male infertility. Acknowledgments. We thank Ms. Frieda Karp for her invaluable technical assistance in performing the flow cytometric evaluations, and Philip Tomashefsky, Ph.D., for statistical analysis. 290 Kaufman and Nagler Testes aspirates Fertility and Sterility
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