Is one testicular specimen sufficient for quantitative evaluation of spermatogenesis?

FERTILITY AND STERILITY Copyright

q,:;

1995 American Society for Reproductive Medicine

Vol. 64, No.2, August 1995 Printed on acid-free paper in U. S. A.

Is one testicular specimen sufficient for quantitative evaluation of spermatogenesis?

Shoshana Gottschalk-Sabag, M.D. *t David B. Weiss, M.D.:j: Nina Folb-Zacharow, M.D.* Zvi Zukerman, M.D.§ Shaare Zedek Medical Center, Jerusalem, and Beilinson Medical Center, The Sackler School of Medicine, Tel Aviv, Israel

Objective: To investigate whether quantitative analysis performed on one testicular specimen is adequate for quantitative evaluation of spermatogenic process. Design: Comparison of quantitative analysis of spermatogenic cell types in testicular cytologic aspirates of various sites of each testis. Setting: In each aspirate, a total of 500 Sertoli cells and cells at each of the spermatogenic stages were identified, counted, and grouped according to cell type. A quantitative cell type index was calculated for each type of cell in each aspirate. Mean cell type indexes then were calculated for each of the cell types in the three aspirates of each patient, and variations of a given sample from its mean were compared. Patients: Azoospermic or severely oligospermic infertile men. Interventions: Fine needle aspiration performed on the upper, middle, and lower poles of each testis. Results: Each of the aspirates showed wide deviations from the mean of the three aspirates for that patient. The deviation ranges of the cell type indexes of each of the spermatogenic stages were as follows: spermatogonia, 0.8% to 200%; spermatocytes, 1.4% to 94.3%; spermatids, 2.9% to 200%; and spermatozoa, 0.7% to 128%. In the majority of the patients, at least one of the three aspirates showed a cell type index score that was statistically different from the others. Conclusions: These results suggest that more than one testicular specimen is needed to evaluate quantitatively the spermatogenic process. Fertil Steril 1995;64:399-402 Key Words: Testis, quantitation of spermatogenesis, fine needle aspiration, male infertility

Analysis of the spermatogenic process is of paramount significance in the evaluation ofthe azoospermic or the oligospermic infertile male (1-8). Recently, the use of testicular fine needle aspiration (FNA) instead of the biopsy has been advocated as a preferable diagnostic technique for this purpose

Received September 7, 1994; revised and accepted February 24,1995. * Department of Pathology and Cytology. t Reprint requests: Shoshana Gottschalk-Sabag, M.D., Department of Pathology and Cytology, Shaare Zedek Medical Center, P.O. Box 3235, 91031 Jerusalem, Israel (FAX: 972-26-513946). :j: Male Infertility Unit. § Department of Andrology. Vol. 64, No.2, August 1995

(9-13). Several methods for quantitative analysis of the spermatogenic process based on FNAs have been suggested (10-12). The FNA technique itself is simple, reliable, and relatively well tolerated, with no complications or adverse effects. The aim of this study was to determine if a single testicular FNA is sufficiently reliable to represent the spermatogenic process occurring in the entire organ. MATERIALS AND METHODS

Fine needle aspirations were performed on one testes of 13 azoospermic or severely oligospermic (sperm count < 1.5 X 10 6/mL) infertile men. The testes were punctured percutaneously with a 23Gottschalk-Sabag et al. Evaluation of spermatogenesis

399

Table 1

Cell Type Indexes of Three Separate FNAs in Each Patient Spermatozoa

Spermatids

Spermatocytes

Spermatogonia

Patient

FNAI

FNA2

FNA3

FNAI

FNA2

FNA3

FNAI

FNA2

FNA3

FNAI

FNA2

FNA3

1 2 3 4 5 6 7 8 9 10 11 12 13

0.49 1.88 0.08 0.05 0.00 1.69 0.46 0.57 2.83 0.21 0.37 0.00 0.65

0.19 1.87 0.16 0.01 0.00 2.94 0.53 0.57 3.73 0.03 2.51 0.01 0.55

0.38 3.33 0.01 0.08 0.00 0.89 0.41 0.24 1.99 0.31 2.02 0.05 0.99

0.13 0.18 0.11 0.04 0.00 0.24 0.02 0.05 0.63 0.06 0.07 0.01 0.25

0.06 0.05 0.04 0.07 0.00 0.85 0.07 0.38 1.10 0.01 0.02 0.00 0.00

0.15 0.28 0.00 0.00 0.00 0.21 0.05 0.14 0.63 0.00 0.31 0.00 0.35

0.33 0.37 0.20 0.08 0.09 0.37 0.15 0.28 0.97 0.13 0.21 0.05 0.59

0.14 0.69 0.21 0.01 0.05 0.77 0.22 0.17 0.89 0.08 0.32 0.00 0.56

0.23 0.38 0.15 0.07 0.13 0.51 0.14 0.15 0.73 0.05 0.96 0.04 0.94

0.02 0.10 0.00 0.01 0.00 0.00 0.09 0.02 0.00 0.00 0.01 0.00 0.04

0.00 0.15 0.00 0.00 0.00 0.11 0.03 0.01 0.15 0.00 0.01 0.00 0.08

0.05 0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.03 0.00 0.11 0.00 0.19

gauge (6/10 mm) needle and aspirated with a 20-mL syringe attached to a syringe holder. Each testis was punctured at three different sites: the upper, middle, and lower poles. The aspirated material from each puncture was smeared on two slides. One was air dried and stained with May-Grunwald-Giemsa stain, and the second was fixed immediately in 95% alcohol and stained with Papanicolaou stain. Smears were examined using light microscopy with a magnification of x250. A total of 500 consecutive spermatogenic and Sertoli cells in each smear were counted. These cells were identified carefully in accordance with their own cell type identification. They were categorized as spermatogonia, spermatocytes, spermatids, spermatozoa, or Sertoli cell groups and each group was quantitated. A cell type index for each type of the spermatogenic cells was then calculated as the ratio of the number of cells of the given spermatogenic cell type to the number of Sertoli cells counted in the aspirate. These indexes were determined for each of the four groups of cells in every aspirate of each individual. RESULTS

The cell type indexes that were determined for each spermatogenic step in each of the three aspirates for each individual are presented in Table 1. Mean cell type indexes were then calculated for each of the cell types in every patient. Table 2 presents cell type indexes range of deviation of any given cell type index from its mean and coefficient of variation (CV). For spermatogonia, this deviation was between 0.8% and 200%; for spermatocytes, it ranged from 1.4% to 94.3%; for spermatids, from 2.9% to 200%; and for spermatozoa, this range of deviation was from 0.7% to 128.5%. In addition the CV is presented in Table 2, as to standardize the effect of variation. It is exceedingly high for many cell type 400

Gottschalk-Sabag et al. Evaluation of spermatogenesis

indexes determined. In 12 of 52 comparisons found in 7 of 13 patients, the differences of at least one of the cell type indexes were statistically significant > 95% from the expected deviations. The range of the "absolute deviation" in each patient was determined and is presented in Table 3. This absolute deviation is defined as the range from the lowest figure to the highest figure of deviation found in any of the four cell type indexes. This deviation represents the actual cell type index variations that occurred among all aspirates taken from the different sites of the same testes. DISCUSSION

Evaluation of testicular spermatogenesis is an important tool in the evaluation of the azooligospermic infertile male. Various qualitative and quantitative methods have been suggested to achieve this goal (1-8, 12, 13). Most of these methods are based upon evaluation of histologic sections taken as a single biopsy from the testis. It has not been determined yet whether a single testicular biopsy specimen can be used to represent the whole testis with regard to the spermatogenic process. Skakkebaek et al. (14) defined a "heterogeneous picture" of the testis as a picture in which one or more seminiferous tubules are involved in spermatogenesis while others are devoid of germ cells. He emphasized that, because the volume of a testicular biopsy is relatively small, the biopsy may not always be representative of the entire testis. In their recent paper, Giwercman and Skakkebaek (15) described the same feature and stated that in some patients one half of the seminiferous tubules may be hyalinized completely and obliterated and the other half may have normal spermatogenic capability. In contrast, Tillinger (16) stated that a testicular biFertility and Sterility

Table 2

Spread of Cell Type Index Deviations From Their Respective Means, CVs, and Their Associated Probabilities* Spermatozoa

Patient

Range

1 45.3 to 7.0 2 41.1 to 20.4 3 93.5 to 2.6 4 74.2 to 2.9 5 6 59.9 to 8.2 7 13.6 to 1.6 47.4 to 23.0 8 9 30.8 to 0.7 10 81.1 to 13.6 11 77.1 to 23.4 12 128.5 to 32.2 36.0 to 10.7 13 Overall 128.5 to 0.7

CV

Spermatids P

Range

42.22 35.55 92.28 72.79

49.2 70.3 <0.05 121.8 100.0 <0.1 200.0 56.25 97.0 12.90 50.3 41.07 100.4 30.46 39.5 169.6 75.20 <0.1 68.50 <0.1 133.7 115.82 <0.05 100.0 100.0 32.05 200.0

to to to to to to to to to to to to to to

18.2 5.4 21.8 2.9 100.0 44.8 13.0 26.0 19.4 69.6 47.5 33.2 24.1 2.9

CV 43.11 70.46 112.48 98.58 173.21 84.13 45.22 90.25 34.18 147.66 117.42 88.22 90.41

Spermatocytes P

Range

42.1 44.3 <0.1 22.4 89.8 <0.05 49.3 39.8 30.1 39.8 15.3 <0.05 54.7 <0.05 94.3 92.8 34.8 94.3

to to to to to to to to to to to to to to

1.4 20.7 7.9 32.2 1.8 7.1 13.2 13.1 2.6 11.2 36.4 24.1 15.4 1.4

CV

Spermatogonia P

41.41 38.41 19.65 78.83 <0.05 48.44 36.81 26.13 35.10 14.15 50.01 <0.1 82.41 <0.05 83.39 <0.05 30.17

Range

CV

P

129.7 88.4 200.0 200.0

to to to to

29.7 14.2 100.0 100.0

117.69 82.24 173.21 173.21

<0.05 <0.05

200.0 119.5 43.9 153.3 24.5 156.2 200.0 88.3 200.0

to to to to to to to to to

100.0 27.4 0.8 53.3 2.4 77.6 100.0 26.2 0.8

173.21 108.46 43.46 134.77 23.41 135.30 173.21 78.59

<0.05 <0.1 <0.1 <0.05

* Values are percentages.

opsy, with very few exceptions, reflects the condition of the whole organ. Fine needle aspiration of the testes is a simple and reliable method of obtaining testicular cytologic elements and is well tolerated by the patients. Recently, methods of quantitative evaluation of spermatogenesis with FNA were introduced (10, 11). We routinely perform three separate punctures, from three different sites, in each testis. Therefore we were able to compare the results of a quantitative analysis of the spermatogenic process determined in each aspirate with the others taken from the same testis. Our results show that in every single patient the quantitative indexes attributed to a specific spermatogenic cell type in each of the three aspirates were quite different. In the majority of patients, the differences of at least one cell type index were statistically significant. Deviations of individual aspirates from the mean ranged up to 94.3% for spermatoTable 3 Range of Absolute Deviations of Cell Type Index From Their Respective Means* Patient 1 2

3 4 5 6 7

8 9 10 11 12 13

* Values

are percentages.

Vol. 64, No.2, August 1995

Range 129.7 88.4 200.0 200.0 200.0 200.0 119.5 100.4 153.3 169.6 156.2 200.0 100.0

to to to to to to to to to to to to to

1.4 5.4 2.6 2.9 1.8 7.1 1.6 0.8 0.7 2.4 23.4 24.1 10.7

cytes, 128.5% for spermatozoa, and as high as 200% for spermatogonia and spermatids. The range of the absolute deviations from the mean, which takes into consideration the lowest and highest figures of deviation in any of the four cell type indexes for each patient found in any of the three aspirates, were very wide. For the entire population of patients, the absolute deviation ranged between 0.7% and 200%. Similar extreme values are seen for the CV, especially for spermatids and spermatogonia. These results suggest that precise evaluation of testicular spermatogenesis cannot be based on a single testicular sample. Testicular tissue is very delicate and vulnerable, and only a very small single specimen is taken when a biopsy is performed. Therefore, we recommend that FNAs from several sites of the testes should be performed when quantitative analysis of testicular spermatogenesis is desired. The main advantage of using the FNA technique is that more than one specimen can be taken harmlessly.

Acknowledgments. The authors thank Zvi E. Kahana, Ph.D., for the statistical analysis and Cathy Hammerman, M.D., for her editorial assistance. REFERENCES 1. Zukerman Z, Rodriguez-Rigau LJ, Weiss DB, Chowdhury AK, Smith KD, Steinberger E. Quantitative analysis ofthe seminiferous epithelium in human testicular biopsies, and the relation of spermatogenesis to sperm density. Fertil Steril 1978;30:448-55. 2. Nelson WOo Interpretation of testicular biopsy. J Am Med Assoc 1953; 51:449-54. 3. Roosen-Runge EC. Quantitative investigation on human testicular biopsies. 1. Normal testis. Fertil Steril 1956;7: 251-61. 4. Mancini RE, Rosemberg E, Cullen M, Lavieri JC, Vilar 0, Gottschalk-Sabag et al. Evaluation of spermatogenesis

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