Quantitative Analysis of The Seminiferous Epithelium In Human Testicular Biopsies, and The Relation of Spermatogenesis to Sperm Density*

FERTILITY AND STERILITY Copyright' 1978 The' American Fertility Society

Vol. 30, No.4, October 1978 Printed in U.s.A.

QUANTITATIVE ANALYSIS OF THE SEMINIFEROUS EPITHELIUM IN HUMAN TESTICULAR BIOPSIES, AND THE RELATION OF SPERMATOGENESIS TO SPERM DENSITY*

ZVI ZUKERMAN, M.D. LUIS J, RODRIGUEZ-RIGAU, M.D. DAVID B. WEISS, M,D, AJIT K. CHOWDHURY, PH,D. KEITH D. SMITH, M,D, EMIL STEINBERGER, M,D.t Department of Reproductive Medicine and Biology, The University of Texas Medical School at Houston, Houston, Texas 77025

Quantitative analysis of the seminiferous epithelium was performed in bilateral testicular biopsy specimens from 14 patients with sperm counts ranging from 0 to 89 million/mI. All Sertoli cells and germ cells within each seminiferous tubule cross-section were counted in all biopsies. Results were expressed either as number of cells per unit length of seminiferous tubule circumference or as number of cells per Sertoli cell. Results were correlated with sperm count (millions per milliliter), total sperm count (millions per ejaculate), and age. A significant correlation between sperm density and germ cell counts was demonstrated. Coefficients of correlation were higher when results were expressed per unit oftubular wall length than when expressed per Sertoli cell. In men with sperm counts below 5 million/ml the number of germ cells in the biopsy was lower than in men with higher sperm counts. Spermiogenesis appeared to be most affected. In this group ofpatients an adverse effect of age on spermatogenesis was .noted. Fertil Steril 30:448, 1978

Microscopic evaluation of testicular biopsies is an important tool in the study of human testicular function. This evaluation is usually performed in qualitative fashion, and results of such examination are expressed in general descriptive terms. Although clear-cut qualitative changes have been described for some conditions affecting testicular function (e.g., Klinefelter syndrome, hypogonadotropic hypogonadism), in a majority of patients a subjective qualitative analysis of the seminiferous epithelium does not· provide sufficient information to arrive at a specific diagnosis. A precise, quantitative evaluation of spermatogenesis may provide information concerning

the etiology and pathogenesis and, in some instances, may serve as a helpful guide to therapy. Several investigators described quantitative methods for evaluation of spermatogenesis in human testicular biopsies. I - B Some were semiquantitative, based on the general appearance of each seminiferous tubule, and did not employ differential cell counts. \.3,6 Clermont 9 in 1963 proposed a quantitative method for evaluation of human spermatogenesis. This method was modified by Steinberger and Tjioe 5 and applied to the study of normal and abnormal testicular biopsies. In this method, cell counts were expressed per unit length of seminiferous tubule circumference:'; Shakkebaek and HellerB suggested later the advantage of expressing germ cell counts per Sertoli cell. The purpose of this study was to investigate the relationship between quantitative analyses of the seminiferous epithelium histology and sperm

Received May 1, 1978; accepted June 2, 1978. *Supported in part by National Institutes of Health Center Grant 5 P50 HD08338. tReprint requests: Emil Steinberger, M.D., Department of Reproductive Medicine and Biology, P.O. Box 20708, University of Texas Health Science Center, Houston, Tex. 77025.

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QUANTITA TIVE ANALYSIS OF SEMINIFEROUS EPITHELIUM

output. Since to our knowledge such correlation has not been published, two mehtods for quantitation of the germinal epithelium (counts expressed per unit of tubular wall length and per Sertoli cell) were used for this purpose and the results were compared. MATERIALS AND METHODS

Fourteen patients, 17 to 38 years of age, with sperm couns ranging from 0 to 89 million/ml and varying degrees of unilateral or bilateral varicocele were selected for the study. The diagnosis of varicocele was established by examination ofthe patients in an upright position. Reflux of blood into the pampiniform plexus was determined by manual palpation, with the patient performing Val salva's maneuver. Varicoceles were graded as small, moderate, or large according to the classification of Dubin and Amelar. 10 Testicular consistency was evaluated subjectively by palpation, and testicular volume was calculated from measurements of testicular diameters utilizing a caliper. Bilateral testicular biopsies were obtained at the time of surgical exploration of the scrotum and varicocelectomy. No evidence of epididymal pathology could be detected in any of these patients. Preoperative evaluation of all patients included multiple semen analyses and measurement of blood hormone levels. All semen specimens were collected by masturbation after a 2- to 4-day continence period. Sperm counts were performed in duplicate utilizing a hemocytometer, and volume was measured in a graduated cylinder. Total sperm count was calculated as the product of the volume of the ejaculate in milliliters and the sperm count (in millions per milliliter). Fresh testicular tissue obtained during surgery was fixed in Cleland's fixative and processed by standard histologic methods. Four-micrometer sections were stained with periodic acid-Schiffhematoxylin and by Masson's trichrome method. The entire histologic section of each biopsy was photographed and a print representing a total enlargement of x100 was made. The tubules were numbered consecutively. The outline of each seminiferous tubule was traced on the print, and its circumference was measured with a highprecision map-measuring instrument. This permitted the calculation of the actual circumference of each tubule. Each tubule was identified in the histologic sections under a microscope. Using a ><100 oil immersion objective, all cells in each seminiferous tubule were identified and counted.

449

The identification was based on the description of the cytologic details of human seminiferous epithelium provided by Clermont,9 and summarized as follows: Sertoli Cell (S). Polymorphous nucleus with folded nuclear membrane; large nucleolus showing spherical core and irregular fringe. Type A Spermatogonia-Dark (Ad). Spherical or ovoid nucleus with deeply stained, dense, homogenous chromatin; one or more clear cavities in the chromatin mass, frequently containing the nucleolus. Type A Spermatogonia-Pale (Ap). Ovoid nucleus with uniform, finely granular chromatin; one or more nucleoli free of chromatin, usually close to the nuclear membrane. Type B Spermatogonia (B). Nucleus contains, in addition to fine chromatin granulation, several heavily stained chromatin masses; mucleolus detached from nuclear membrane. Preleptotene Spermatocyte (PL). The spherical nucleus contains well-stained chromatin granulation of uniform size which is distributed throughout the nuclear sap; one or more nucleoli are seen near the center of the nucleus. Leptotene Primary Spermatocytes (L). Chromatin granules changed to filaments. Zygotene Primary Spermatocytes (Z). Presence of coarser chromatin filaments. Pachytene Primary Spermatocytes (P). Chromatin filaments shortened and thickened, to give characteristic pachytene appearance. Spermatids a (Sa). Spherical nucleus which contains a few heavily stained granules in addition to a homogenously pale-stained chromatin; the nuclear surface is depressed by a· small acrosomic vacuole. Spermatids b (Sb). The chromatin is more diffuse and more intensely stained; the chromatin flakes have disappeared; the nucleus has lost its perfectly spherical shape to elongate slightly. Spermatids c (Sc). The nucleus is more pointed; the chromatin is condensed and heavily chromophilic. Spermatids d (Sd). The chromatin has undergone further condensation; the nucleus is further reduced in size and has assumed its definitive shape. Tubules cut at oblique angles showing no lumina were disregarded. All cell counts were performed by the same individual (one of the authors) and were expressed as (1) number of cells per unit length (31 ILm) of circumference of the seminiferous tubule and (2) number of germ cells

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TABLE 1. Clinical Information of the Study Population Testis volume Patient

Age

12 13 14

24 30 26 32 28 17 27 29 22 28 28 38 35 23

Mean± SE

27.8 ± 1.4

1 2 3 4 5 6 7 8 9 10 11

Varicocele site and degree"

L-s L-m L-m L-s L-I L-s L-m; R-s L-s; R-s L-m; R-s L-I; R-s L-I; R-s L-s; R-s L-s L-m; R-s

Left

Right

Sperm countb

Total sperm countb

ml

ml

x IO'lml

x IO'lejaculate

13.1 10.1

23.6 15.3

13.1 5.6

13.1 23.6

41.8 23.6 3.5 8.4 23.6 6.3 9.0 13.0

41.8 23.6 14.7 23.6 23.6 6.3 9.0 20.0

75.7 0 22.0 41.0 2.2 89.0 16.0 19.0 1.8 0.3 4.3 0.3 0.3 0.6

235.3 0 55.0 21.8 5.4 160.2 5.7 49.4 5.6 0.6 4.7 0.8 0.6 1.8

14.2 ± 3.2

19.7 ± 2.8

19.4 ± 7.8

39.0 ± 19.0

ilL, Left side; R, right side; s, small; m, moderate; I, large. "Means of multiple counts.

per Sertoli cell. The counts of leptotene and zygotene primary spermatocytes were pooled. Spermatids were grouped as early spermatids (Sa + Sb) and late spermatids (Sc + Sd). The following indices were calculated from the cell counts expressed both per "unit" of circumference length of the seminiferous tubule and per Sertoli cell: (1) total spermatogenesis-sum of all germ cells; (2) total spermatogonia (Sg)-sum of type A and type B spermatogonia; (3) total spermatocytes (Sy)sum of pre leptotene, leptotene, zygotene, and pachytene spermatocytes; and (4) total spermatids (Si)-sum of early (Sa + Sb) and late (Sc + Sd) spermatids. For the statistical evaluation of the results a linear regression analysis was performed and the correlation coefficients and t values were calculated by standard techniques. Patients were also grouped according to sperm count, and the groups were compared by one-way analysis ofvarianceY These statistical analyses were performed on a Hewlett-Packard 9830A desk-top computer.

RESULTS

Age, varicocele site and degree, testicular volume, sperm count, and total sperm count for each patient are summarized in Table 1. The mean sperm count and total sperm count were 19.4 million/ml and 39.0 million/ejaculate, respectively. In eight patients, the mean sperm count was below 5 million/ml. No differences in sperm count were detected when patients were grouped by degree of varicocele. The mean vol-

ume of the left testes was significantly lower (P < 0.05) than that of the right. The number of seminiferous tubule crosssections counted in each biopsy, and the differential cell counts expressed per "unit" (31 ILm) of tubular wall length are shown in Table 2. The number of seminiferous tubule cross-sections counted were similar for all biopsies, with a minimum of 32. Steinberger and Tjioe 5 demonstrated that a minimum of 25 tubules should be counted to obtain accurate results. A variation in number of Sertoli cells per "unit" was noted, both between patients and between right and left testes for each patient. However, as a group no significant difference in Sertoli cell number could be detected between right and left testes. Similarly, no significant differences in germ cell counts could be demonstrated between right and left testes, either when expressed per "unit" of tubular wall length or per Sertoli cell (Table 3). The total number of spermatogonia, spermatocytes, and spermatids represented approximately 19%, 38%, and 44% of total germ cells, respectively. A significant correlation between each cell type count and either the preceding or following cell type count was observed (P < 0.01 or P < 0.001). Significant correlations between sperm count and total spermatogenesis, total spermatids, and late spermatids expressed per "unit" were observed (Fig. 1). The highest t value and coefficient of correlation were observed for the last steps of spermiogenesis (coefficient of correlation 0.77, P < 0.01). Similar correlations were also detected for total sperm count. The coefficient of correla-

QUANTITATIVE ANALYSIS OF SEMINIFEROUS EPITHELIUM

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tion between sperm count and total sperm count was 0.9 (P < 0.001). When correlations between sperm density and germ cell counts expressed per Sertoli cell were analyzed, the correlation with late spermatids was significant. However, the coefficient of correlation (0.67) was lower than when expressed per "unit" (0.77). No significant correlation of total spermatogenesis per Sertoli cell or total spermatids per Sertoli cell and sperm density could be demonstrated. An adverse effect of age on sperm count was observed in this group of patients (Fig. 2). The coefficient of correlation was -0.54, significant at the 0.05 level. The ratios of type A spermatogonia-dark to late spermatids, total spermatocytes to total spermatids, and early spermatids to late spermatids were significantly correlated with age (Fig. 2), demonstrating that in this group of patients aging was associated with a progressive decline of spermiogenesis. When the patients were grouped according to sperm count (below and above 5 mill ion/ml), significant differences were demonstrated for each cell count expressed per "unit," except for Sertoli cells and pre leptotene primary spermatocytes (Fig. 3). Significant differences between the two groups were also observed for total spermatogenesis, spermatogonia, spermatocytes, and spermatids. The differences were not uniform for each cell type. Total spermatogonia, spermatocytes, and spermatids per "unit" in the group of patients with sperm counts below 5 million/ml represented 67%, 76%, and 31% of the counts in the group with higher sperm count. Late spermatids were maximally decreased (23%). The total number of germ cells per unit oftubular wall length in the group with lower sperm counts represented 52% of that in the second group. When similar comparisons between these two groups of patients were performed for cell counts expressed per Sertoli cell, differences for several cell types and total spermatocytes could not be demonstrated (Fig. 3). In addition, for those differences that were significant, the t value was lower than when the cell counts were expressed per "unit." Although most germ cell counts were lower in the group with sperm count below 5 million/ml than in the group with higher sperm counts, a relative accumulation of type A spermatogonia, total spermatocytes, and early spermatids in respect to late spermatids was observed in the first group (Fig. 4).

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October 1978

ZUKERMAN ET AL.

TABLE 3. Differential Cell Counts of the Germinal Epithelium Expressed per Sertoli Cella Ap

Ad

B

Patient

1 2 3 4 5 6 7 8 9 10 11

12 13 14 Mean SE

L +Z

PL

P

Sa + Sb ----L R

L

R

L

R

L

R

L

R

L

R

L

R

0.5 0.2 0.2 0.2 0.2 0.1 0.2 0.4 0.2 0.1 0.2 0.2 0.0

0.5 0.1 0.2 0.2 0.2 0.1 0.1 0.7 0.2 0.3 0.1 0.2 0.1 0.1

1.1 0.6 0.6 0.4 0.4 0.5 1.0 0.7 0.8 0.6 0.4 1.0 0.2

1.1 0.7 0.8 0.6 0.3 0.5 0.9 1.4 0.8 0.9 0.4 0.9 0.5 0.3

0.1 0.1 0.0 0.0 0.0 0.0 0.1 0.1 0.1 0.0 0.0 0.0 0.0

0.1 0.1 0.0 0.1 0.0 0.0 0.1 0.1 0.1 0.1 0.0 0.0 0.0 0.0

0.3 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.3 0.1 0.1 0.3 0.1

0.2 0.2 0.1 0.1 0.0 0.1 0.2 0.2 0.2 0.1 0.1 0.4 0.1 0.0

0.2 0.1 0.2 0.1 0.0 0.1 0.2 0.3 0.2 0.1 0.1 0.1 0.7

0.2 0.0 0.1 0.1 0.0 0.1 0.1 0.2 0.2 0.1 0.1 0.2 0.1 0.0

2.5 1.2 1.3 1.0 0.5 1.4 1.8 1.6 2.8 0.7 1.1 2.9 0.8

3.1 1.7 1.4 2.7 0.4 1.3 1.2 2.9 2.7 0.9 1.0 2.8 1.2 0.9

3.9 0.8 2.0 1.0 0.8 1.0 2.9 1.3 1.8 0.4 0.2 0.5 0.4

0.2 0.0

0.2 0.0

0.7 0.0

0.7 0.1

0.1 0.0

0.1 0.0

0.2 0.0

0.2 0.0

0.2 0.1

0.1 0.0

1.5 0.2

1.7 0.3

1.2 0.3

Total germ cells

Se + Sd L

R

L

R

4.2 1.1 1.9 0.8 0.4 0.9 1.7 2.9 1.6 0.6 0.1 0.4 1.1 0.9

2.4 0.1 0.6 0.8 0.6 1.2 1.9 1.5 1.1 0.1 0.1 0.6 0.1

3.6 0.6 0.9 2.8 0.5 1.0 1.0 1.9 0.9 0.2 0.0 0.0 0.7 0.3

10.8 3.1 5.0 3.5 2.7 4.3 8.2 6.0 7.4 2.0 2.1 5.7 2.1

13.0 4.5 5.5 8.5 1.9 3.9 5.1 10.3 6.6 3.2 1.7 4.9 3.7 2.5

1.4 .3

0.8 0.2

1.0 0.3

4.8 0.8

5.4 0.9

a L, Left testis; R, right testis; Ad, type A spermatogonia-dark; Ap, type A spermatogonia-pale; B, type B spermatogonia; PL, preleptotene spermatocytes; L + Z, leptotene and zygotene spermatocytes; P, pachytene spermatocytes; Sa + Sb, spermatids a + b; Sc + Sd, spermatids c + d.

DISCUSSION

Two methods have been proposed for the quantitative analysis of the seminiferous epithelium in human testicular biopsies. The method described by Steinberger and Tjioe 5 is based on the measurement of the circumference of each seminiferous tubule in a biopsy and identification of each cell type to be counted. All cell counts in this method are expressed per unit oftubular wall length. The method reported by Skakkebaek and Heller 8 is based on the determination of the germ cell to Sertoli cell ratio. These authors state that the variation in this ratio between individuals is relatively small. However, they also noted that in subfertile individuals this ratio may vary and thus preclude the clinical application of their

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method. Indeed, several investigators 5. 12 reported a high interindividual variation in Sertoli cell number and concluded that the stability of the Sertoli cells cannot be assumed for the human testis. Our results support this conclusion, since a relatively high variation in Sertoli cell number was observed, both between individuals and between right and left testes of the same patient. When correlations between sperm output and quantitative analysis of spermatogenesis performed by the two methods were compared, higher coefficients of correlation were noted when cell counts were expressed per unit of tubular wall length. When all counts from patients with sperm counts below 5 million/ml and above 5 million/ml were compared, the method utilizing tubular wall length as the basis for reference

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FIG. 1. Correlation between sperm count and total spermatogenesis, number of spermatids, and number of late spermatids (Sc + Sd) per "unit."

453

QUANTITATIVE ANALYSIS OF SEMINIFEROUS EPITHELIUM

Vol. 30, No.4

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FIG. 3. Comparison of quantitative analysis of spermatogenesis expressed per "unit" or per Sertoli cell, for patients with sperm counts below and above 5 million/ml (S, Sertoli cell; Ad, type A spermatogonia-dark; Ap, type A spermatogonia-pale; B, type B spermatogonia; PL, preleptotene primary spermatocyte; L, leptotene primary spermatocyte; Z, zygotene primary spermatocyte; P, pachytene primary spermatocyte; Sa + Sb, early spermatids; Sc + Sd, late spermatids; Sg, total spermatogonia; Sy, total spermatocytes; Si, total spermatids; Total, germ cells; *. P <0.05; ** ,P < 0.01; ***, P < 0.001).

454

October 1978

ZUKERMAN ET AL .

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FIG. 4. Comparison of ratios between germ cell counts for patients with sperm count below and above 5 million/ml (A/after, ratio of counts of type A spermatogonia to the sum of the counts of type B spermatogonia, spermatocytes, and spermatids; Sy/Si, ratio of counts of spermatocytes and spermatids; Sa + Sb/Sc + Sd, ratio of counts of early and late sperm at ids; Si/before, ratio of counts of spermatids to the sum of the counts of spermatogonia and spermatocytes; Sc + Sd/before, ratio of the count of late spermatids to the sum of the counts of spermatogonia, spermatocytes, and early spermatids).

demonstrated significant differences in each cell type count. Utilizing the germ cell/Sertoli cell ratio no differences in type A spermatogonia-pale or any of the primary spermatocytes could be demonstrated between the two groups of patients. On the basis of these findings it appears appropriate to conclude that, for evaluation of spermatogenesis in testicular biopsies from subfertile men, utilization of tubular wall length as a point of reference is probably a more precise method. Although the presence of a varicocele was common to all patients studied, no correlation of degree of varicocele to spermatogenesis or sperm density was demonstrated. These findings are in agreement with the recent report by Agger and Johnsen,13 who evaluated spermatogenesis in testicular biopsies from 81 patients with varicocele. Although these results suggest lack of relation of varicocele size to the disturbance of spermatogenesis found in some of our patients, it has been noted by other authors that the detrimental effect of varicocele may be related to its presence and duration, but not to its size. 14 Indeed, a significant adverse effect of age on sperm density was noted for our group of patients. The counts oflate spermatids were most affected by age. A relative accumulation of spermatogonia, spermatocytes, and early spermatids was noted to be correlated with age. Although an adverse effect of age on testicular function has been demonstrated,15. 16 since the presence of varicocele is established at the time of puberty,14 the effect of age on spermatogenesis noted in this study could be explained by time-effect of the varicocele, or by ac-

celeration of the aging process in the presence of a varicocele. A lack of correlation between results of semen analysis and histologic evaluation of spermatogenesis was reported by several investigators. 2 • 17 Utilizing a semiquantitative method, Mannion and Cottrell 3 and Agger and Johnsen 13 demonstrated correlation between sperm output and various patterns of the seminiferous epithelium. In this study, significant correlations of sperm count and total sperm count to total spermatogenesis, total number of spermatids, and, most significantly, with the number of mature spermatids were demonstrated. Although the number of spermatozoa in the ejaculate reflects the entire process of spermatogenesis, it is not surprising to observe a higher degree of correlation between sperm output and number of late spermatids than with the number of prehaploid cells. It should be noted that this correlation was demonstrated for both oligospermic individuals and men with higher sperm counts. In subfertile men with varicocele a spermatogenic arrest at the primary spermatocyte or spermatid levels has been suggested to be common. IS In our group of patients, quantitative analyses of spermatogenesis revealed a decreased count of all cell types in men with sperm counts below 5 million/ml compared with men with higher sperm counts. Patients were separated into two groups (sperm count below and above 5 million/ml) on the basis of a recent report suggesting 5 million/ml as the borderline between fertility and subfertility.19 The decrease in germ cell

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QUANTITATIVE ANALYSIS OF SEMINIFEROUS EPITHELIUM

count was not uniform for each cell type in patients with low sperm count; the counts of early and late spermatids were maximally suppressed. Consequently, in some patients a relative accumulation of primary spermatocytes wasobserved, and in others early spermatids were relatively increased. Although a maximal decrease in late spermatid count was common to all oligospermic patients, it should be emphasized that the absolute count of all other cell types, including spermatogonia, was also suppressed. In conclusion, quantitative analysis ofspermatogenesis in testicular biopsies from subfertile men permits localization ofthe lesion to a specific cell type in the seminiferous epithelium. Since different stages of the process of spermatogenesis are under different hormonal control,20 this localization may provide valuable information regarding the etiopathogenesis and serve as a guide to therapy. Acknowledgment. We wish to express thanks to Dr. H. Edward Grotjan, Jr., for his kind assistance in the statistical evaluation of the results. REFERENCES 1. Nelson WO: Interpretation of testicular biopsy. JAMA 151:449, 1953 2. Roosen-Runge EC: Quantitative investigation on human testicular biopsies. I. Normal testis. Fertil Steril 7:251, 1956 3. Mannion RA, Cottrell TLC: Correlation between testicular biopsy and sperm count. J Urol 85:953, 1961 4. Mancini RE, Rosemberg E, Cullen M, Lavieri JC, Vilar 0, Bergada C, Andrada JA: Cryptorchid and scrotal human testes. I. Cytological, cytochemical and quantitative studies. J Clin Endocrinol Metab 25:927, 1965 5. Steinberger E, Tjioe DY: A method for quantitative analysis of human seminiferous epithelium. Fertil Steril 19:960, 1968 6. Johnsen SG: Testicular biopsy score count-a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones 1:2,1970

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