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Quantitative Investigations on Human Testicular Biopsies
FERTILITY AND STERILITY VOLUME
8
MAY-JUNE,
1957
NUMBER
3
Quantitative Investigations on Human Testicular Biopsies II. Infertility and Other Conditions
Edward C. Roosen-Runge, M.D., Eve Marberger, M.D., and Warren 0. Nelson, Ph.D.*
IN
THE PRECEDING PAPER9 (hereafter referred to as paper I) 26 normal human testes were analyzed with respect to the volume proportions of their histologic elements, and some standards for the evaluation of testicular biopsies were established. Chalkley's method was used, and described and discussed in detail. As a result of this study it was decided to investigate the volume proportions of testicular components in various abnormal conditions which had never previously been explored quantitatively. Two major objectives were sought: ( 1) to define certain abnormal states of sperma-
From the Departments of Anatomy, University of Washington School of Medicine, Seattle, Wash., and State University of Iowa, College of Medicine, Iowa City, Iowa. Aided by grants from the National Science Foundation (G-516) and the U.S. Public Health Service (RG-1778). Thanks are due to Mrs. J. A. Gromons for able technical assistance. • Present address: Rockefeller Institute, New York, N. Y. 203
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to genesis in quantitative terms, and ( 2) to ascertain· the degree of applicability of the method to clinical investigations. MATERIAL AND METHOD
A total of 180 testicular biopsies from 125 men were analyzed. Bilateral biopsies were available in 42 cases. The specimens were wedges of tissue obtained by experienced operators and preserved immediately in Bonin's or Stieve's solution. Sections were stained with various trichrome stains and in some cases with Gomori's Aldehyde-Fuchsin method. The cases fell into several categories, which will be treated separately below. The classification of the material is indicated in the first 3 columns of Table l. Clinical diagnosis and evaluation were minor factors in the selection of cases for this study. The main objective was to obtain a fairly large number of well-preserved biopsy specimens from a variety of cases. Only specimens were used which showed a fair number of germ cells. Cases with germinal cell aplasia, complete spermatocytic arrest, or severe peritubular fibrosis were not studied. The diagnosis of "infertility" was made in men who had been unable to effect a conception during a period of at least 1 year-in most instances it was several years. In the large majority of cases, the wives had been examined and pronounced potentially fertile. The average ages of the first 4 groups of cases of Table 1 were almost identical. They ranged from a mean of 27.8 years for the nitrofuran cases, to 30.0 years for cases of infertility with oligospermia or normospermia. In the miscellaneous cases the average age was significantly greater: 43 years. The average age of the cases of infertility without sperm counts could not be determined. Chalkley's method will not be described here, as extensive descriptions of the procedure have recently been published.'· 10 It appears necessary, however, to explain once again some of the terms used in this and the previous paper. The term "space" is used for the structures in the seminiferous tubules which could not be identified, regardless of whether or not they appeared to be parts of cells or artifacts. The term "lumen" does not seem to need a specific explanation, but it should be kept in mind that in biopsy specimens the lumen of the tubules is often filled with cells in addition to a fine coagulum. In these cases the cells were recorded in the same way as
TABLE 1.
Classification of Cases and Means of Volume Proportions of Testicular Components Compared with Normal Value~ Testicular components
Condition
No. patients
Normal (standard error of the mean in p"a.rentheses) 26
No. biopsies
Basement membrane and tunica
propria
Leydig cells
Spermatids and Spermato- Spermato- spemwcytes tozoa gonia
14.4 (0.47)
9.1 (0.36)
SertoU ceUs
17.4 (0.38)
Lumen
Total germ cells (including abnormals)
10.6 (0.59), 9.8 for 24
32.0 (0.68)
30
9.1 (0.33)
3.1 (0.23)
7.8 (0.20)
15
23
9.3
2.2
7.4
13.3
7.3
17.8
8.5
28.6
56
80
10.4
3.0
7.5
10.6
4.6
20.7
8.1
23.7
14 14 9 17
21 21 12 23
9.4 8.9 9.9 11.5
2.1 2.7 1.5 2.3
6.9 7.1 7.9 6.5
12.0 13.0 10.4 12.3
7.2 7.2 5.2 3.2
18.8 20.2 19.7 20.3
6.1 8.1 8.4 8.9
26.8 27.8 24.5 23.3
biopsies
Infertility with azoospermia Infertility with oligo- or nonnospermia Nitrofuran treatment Before Mter Infertility, no sperm count Miscellaneousa cases
a Four patients with schizophrenia, 2 with Addison's disease, 2 with "delayed puberty," 2 with hydrocele, 2 over 70 years old, 1 each with myxedema, sickle cell anemia, gyr.ecomastia, other testicle cryptorchid and one without diagnosis.
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those in their natural positions; the coagulate was recorded as lumen. Additional definitions of terms may be found in paper I. INFERTILITY WITH AZOOSPERMIA
It can be assumed that all cases of this group had some obstruction of the seminal passages, as the seminiferous tubules appeared to produce some normal, mature spermatozoa in all of them. The mean volumes of this group are recorded in Table 1. In most instances, differences from the normal standards are slight. The most significant deviation occurs in the volume of the total germ cells, which is 28.6 per cent, as compared with the normal mean of 32 per cent. More than half of this difference is accounted for by a comparatively low volume of spermatids and spermatozoa. The much smaller difference in the volume of the spermatogonia is statistically not significant. In general, the azoospermic patients show fewer deviations from the normal than any other group investigated. A detailed analysis of the 15 cases ( 23 testes) showed that 8 ( 13 testes) were within normal range in all values. In 2 cases 1 testis was normal, the other showed a decrease of the total germ cell volume to just below the normal range ("hypoplasia''). In 2 other cases both testes showed a decrease in total germ cell volume. In these 4 testes the volume of spermatids and spermatozoa was dispropmtionately low, probably indicating some "arrest" of spermatogenesis at the spermatid stage. In another 2 cases ( 2 testes) severe decreases in total germ cell volume were recorded ( 14.6 and 20.7 per cent) and, in addition, the volume of spermatids and spermatozoa was below 50 per cent of the lowest value in the normal series. The data confirm quantitatively what has been known for some time, that spermatogenesis is not appreciably affected in men with obstruction of the ducts. Nelson' has presented a series of 21 cases with blocked or absent efferent ducts. In 8 cases the spermatogenic pattern was inferior, but this did not appear to be related to the defect in the seminal passages. In the present series, one third of the cases showed inferior spermatogenesis and in 2 cases a severe degree of deficiency was present. This proportion appears to be no greater than in a randomly selected series of men who have no obvious defects of reproduction (see nitrofuran cases below). On the other hand, it should be emphasized that the control series (paper I) was not randomly selected but carefully chosen for "normality" of the testicular tissues.
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TESTICULAR BIOPSIES: II
The view is often expressed in pathologic practice that widened lumina of seminiferous tubules are frequently found in patients with obstruction of the ducts. Our data do not support this view. Only one specimen showed a somewhat increased relative volume of the lumen and the average value for lumen is comparatively low. INFERTILITY WITH OLIGOSPERMIA AND NORMOSPERMIA In the average, the cases in this group show a more inferior quantitative picture than the preceding category. Basement membrane and tunica propria are increased in volume, the value for total germ cells is low, the TABLE 2.
Correlation of Sperm Counts and Relative Volume of Total Germ Cells in the Testis in 80 Biopsy Specimens fro~:n Patients with ''Infertility''
Counts
From "very few" to . 0.9mil./cc. 1-10 mil.jcc. 11-50 mil./cc. 51-105 mil./cc.
No. specimens
Relative volume of total germ cells ln %af testis volume
17 26 26 11
16 26 26 26
volume of spermatids and spermatozoa is greatly disproportionately de" creased, and the Sertoli cells show higher than normal values. Both testes were found to be within normal limits in 7 cases. In 2 cases one testis was normal, the other abnormal to a slight extent. Of 80 testes, 56 were definitely abnormal. Of these, 40 had a relative volum~ of spermatids and spermatozoa below 5 per cent (the lowest limit of "normal"). In 21 cases only the spermatids and spermatozoa were decreased, indicating arrest at the early spermatid level, in 6 cases the spermatogonia alone appeared to be of normal volume (arrest at early spermatocyte level), and in 9 cases the volumes of all germ cell generations were proportionately decreased (proportional hypoplasia). An increase in basement membrant: and tunica propria was recorded in 17 cases; the highest value was 18.1 per cent. Sertoli cell volume was increased to an average of 20.7.per cent in the 40 specimen,s and was associated with subnormal volumes of spermatids and spermatozoa in 70 per cent of these cases. Each of the 56 patients in this group had at least orie sperm count, the
TABLE 3.
Total sperm count Case no. (millions) la. R
L lb. R
L 2a. L 2h.R Sa. R L Sb.R L 4a. R L 4b.R L 5a. R 5b.R L 6a. L 6b.R L 7a. R L 7b.R L Sa. R
30 00
No count
..
40 No count
29 00
No count
..
46
..
No count
..
547• 503•
..
No count
No count 00
480
..
No count 00
223
Relative Volumes of Testicular Components in Cases (a) before and (b) after Treatment with Nitrofuran
Basement membrane Spermato~
Spermat~
propria
Leydig cells
gonia
cytes
8.6 9.9 6.4 9.6 7.4 7.1 11.7 9.9 11.2 7.1 7.7 10.7 7.9 8.3 7.0 10.6 8.7 11.0 5.6 8.1 8.9 8.1 9.8 7.6
0.4 1.4 0.7 1.4 2.3 3.0 1.0 2.3 1.4 1.3 1.4 1.3 4.4 1.1 2.6 2.7 3.9 3.3 4.8 4.0 1.4 4.1 2.9 3.3 4.0
5.7 7.7 8.5 8.3 4.0 6.6 4.7 7.1 6.0 8.1 6.3 7.4 5.0 5.9 6.9 7.0 7.1 5.6 5.4 6.7 7.7 7.5 10.3 9.6 7.7
and tunica
9.4
15.0 15.0 13.3 20.7 5.7 9.6 11.6 12.1 10.7 13.4 14.4 13.0 13.4 14.7 10.1 9.4 12.1 11.1 11.7 14.6 12.2 11.9 15.3 15.0 10.6
Spermatid$ Abnormal and germ spermatozoa cells
6.7 8.7 7.0 7.6 2.3 6.4 7.6 8.9 6.3 6.9 11.0 9.7 10.4 5.3 6.4 7.7 6.9 5.0 7.0 8.4 8.7 7.1 8.6 8.0 7.1
1.7 1.6 0.7 0.6 0.3 1.3 0.4 0.3 0.1 0.4 0.7 0.7 0.3 0.9 0.4 1.1 1.0 1.0 0.6 0.1 1.1 0.6 0.6 0.4 1.0
Total germ Sertoli cells
Lumen
cells
18.9 15.8 16.9 18.0 16.7 21.8 20.7 15.3 20.9 22.7 17.4 21.7 23.6 20.7 23.1 18.1 25.0 19.3 17.4 24.2 17.2 15.3 18.8 22.3 17.1
2.4 9.6 8.2 6.6 7.0 11.6 10.1 10.4 6.1 8.9 9.1 5.2 11.9 5.0 4.6 6.3 4.3 3.1 11.4 8.1 9.7 11.8 8.3 11.4 6.5
29.1 33.0 29.5 37.2 12.3 23.9 24.3 28.4 23.1 28.8 32.4 30.8 29.1 26.8 23.8 25.2 27.1 22.7 24.7 29.8 29.7 27.1 34.8 33.0 26.4
L 8b. R L 9a. R L 9b. R lOa. R IOb;L lla. R lib. L 12a. L 12b. R 13a. R L l3b.L 14a. L l4b.R a
.. .. 172 ..
No count
No count No count No count
340• 100 730 240 No count
..
No count
853 No count
9.7 8.1 8.0 10.3 15.9 10.0 8.9 9.7 10.0 8.1 8.0 ll.3 9.1 8.3 13.7 7.7 10.7
Count done 1--S weeks after biopsy.
1.0 2.3 l.l 1.3 1.0 2.1 2.3 3.7 4.6 3.7 2.0 3.7 l.l 2.4 1.9 2.6 4.6
6.0 8.2 6.1 9.4 7.7 7.4 6.3 5.3 7.1 7.6 9.9 5.7 6.4 7.0 8.1 6.0 6.6
10.4 13.3 15.3 14.9 12.1" 15.6 12.4 10.7 10.9 10.2 12.7 10.2 14.3 ll.3 12.9 11.0 10.3
6.9 9.8 8.9 11.0 4.7 7.9 8.0 5.7 4.0 7.7 9.4 5.1 5.6 4.1 3.1 7.4 7.3
1.6 0.7 1.0 0.6 0.7 0.6 0.4 0.1 0.4 0.1 0.3 0.7 0.9 0.6 0.1 0.4 0.1
2l.l 18.2 24.1 16.8 16.9 16.7 22.0 18.4 28.7 24.6 19.0 17.1 15.3 14.7 16.7 21.4 18.4
5.9 9.0 8.3 6.6 3.4 10.0 9.2 7.0 8.4 9.3 14.6 3.9 9.7 9.2 7.6 8.1 6.7
24.9 32.0 31.3 35.9 25.2 31.2 27.1 21.8 22.4 25.6 32.3 21.7 27.2 23.0 24.2 25.7 24.3
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majority had two or more. The counts were done in various laboratories and were not rigidly comparable nor reliable. Therefore, only a rough estimate of the correlation between volume measurements and sperm counts is attempted in Table 2. It appears that there is no appreciable correlation with the sperm counts above 1 million fcc. of semen but that very low counts are usually associated with a severe decrease in germ cells. Analysis of the individual cases confirmed this impression and showed that all germ cell volumes below 19 per cent had counts of 25 millionjcc. or less. Sixteen specimens ( 20 per cent) fell into this category. While this definite positive correlation is of real significance, the lack of correlation for counts ranging from 1 to 105 million/cc. is probably not significant in view of the technics by which some of the counts were obtained. In addition, the counts were done in ahnost all cases either at the time of biopsy or weeks to months before biopsy. CASES BEFORE AND AFTER TREATMENT WITH NITROFURAN The specimens in this group were obtained in the course of an investigation by Nelson and Bunge' on the effect of therapeutic doses ( 5-10 mg./kg.) of Furadantin on human spermatogenesis. It had been shown earlier' that the various nitrofurans have the capacity of arresting spermatogenesis at the spermatocyte stage in the rat. In the present paper only the quantitative analyses of human testicular biopsies will be reported. At least two biopsies, taken at an interval of about 2 weeks, were available in every case, and in 5 individuals two successive biopsies were taken from each testis. The data are shown in detail in Table 3, which may serve as an example of the type of information which forms the basis for all results presented in this paper. The mean values of the group are recorded in Table 1. No significant quantitative differences in volume proportions were observed after treatment with nitrofuran. This essentially confirms Nelson and Bunge's impressions. There are in this series 15 testes with deficient spermatogenesis, and the average relative volumes are slightly more abnormal than in the group with azoospermia. (All subnormal biopsies came from inmates of a prison who volunteered for the experiments.) Once again no correlation could be demonstrated between sperm counts and defects in spermatogenesis, although the counts in this group were relatively well controlled and their timing with respect to the biopsies was favorable for the establishment of correlations. In 5 cases high sperm counts
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were associated with subnormal germ cell volumes. In case 5, for example, the two biopsies of the right testis showed subnormal volumes of all germ cell generations and the total germ cell volume of the left testis (after treatment) was in the lowest normal range. Sperm counts, however, which were done 5 and 2 days before and 5, 9, 16, and 60 days after the first biopsy all showed high normal values. Results were similar in cases 11, 12, and 14, and in 1 case of the normospermic infertility group. In every one of these 5 cases the quantitative deficiency consisted of a proportional decrease in all germ cell generations and no "arrest" was observed. The findings might be explained by assuming a large total volume of the testes in these cases. Exceptionally large testes might well produce a high sperm count even if the average productivity of the seminiferous epithelium is low. How similar are biopsies from the same individual or from the same testis after 2 weeks? Our results appear to indicate- that they are in general quite similar, particularly if the same testis is analyzed. Usually the two testes of the same individual are also similar, but exceptions are seen, for instance, in cases 2 and 12. Among the 42 cases in which simultaneous bilateral biopsies were done, 10 differed to such a degree that one testis was definitely abnormal while the other one was normal. Charny has emphasized "startling similarity" of biopsy specimens removed before and after treatment from the same testis. From our observations we can state that the similarity is usually more impressive qualitatively than quantitatively. MISCELLANEOUS CASES Diagnoses and volume proportions of these cases are summarized in Table 1 and footnote. Most patients of this group suffered from definite diseases and showed in general far more deficient spermatogenesis than the patients with complaints of infertility. It must, however, be kept in mindthat the average age of this group was much higher than that of any of the other categories. · Three out of 4 cases with schizophrenia showed relatively low volumes of spermatids and spermatozoa (indication of "arrest"),'but in only 1 case was the total volume of germ cells below normal limits. In general, the biopsies from schizophrenic patients were among the most normal of the miscellaneous series.
One of the 2 cases with Addison's disease had a quantitatively normal biopsy (sperm counts 110-140 millionjcc.). The other (no sperm counts)
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had a greatly decreased germ cell volume with a very low volume of spermatids and spermatozoa ( 1.9 per cent). The Leydig cell volume was entirely normal in both cases. Subnormal germ cell volumes were found in the biopsy specimens from 2 cases with the diagnosis of "delayed puberty." In one case neither spermatids nor spermatozoa were present; the spermatogonial volume was 4.9 per cent, the spcrmatocytic volume 1.1 per cent. The other case presented a picture of less severe arrest with 2.9 per cent spermatids and spermatozoa. In 2 cases with hydrocele the basement membrane and tunica propria were greatly increased and the volume proportions of the germ cells indicated arrest, with normal spermatogonial volume. Biopsies from 2 men, 70 and 74 years old, showed a general decrease in all generations of germ cells, with the most severe decline in the spermatids and spermatozoa ( 0.7 and 3.2 per cent) and slight increases in the basement membrane and tunica propria. A case of myxedema also had a general and severe decrease in germ cells and an increase in basement membrane and tunica, but this was one of the rare cases in which spermatids and spermatozoa appeared in the normal proportion although the total volnme of germ cells was only 10 per cent. ' A patient with sickle cell anemia had a quantitatively normal biopsy specimen. An individual with gynecomastia ( 47 years old) showed low genn cell volumes in both testes ( 18.6 and 17.9 per cent) with disproportional . decrease in spermatids and spermatozoa and an increase in basement membrane and tunica propria. In the scrotal testis of an individual ( 19 years old) who had one cryptorchid testis, severe disproportional decrease of spermatids and spermatozoa was recorded but .no other abnormalities were found. COMPARISON OF RESULTS IN TWO DIFFERENT LABORATORIES The results presented in the preceding sections of this paper are based entirely on counts made in one laboratory (Washington). Table 4 permits a comparison of data from two laboratories who worked independently with Chalkley's method on the identical preparations from 92 randomly selected cases. The comparison adds to our information concerning the reliability of the method when applied to testicular biopsies. It also con£rms the crucial importance of the criteria of identification. The greatest difference in the data obtained by the two laboratories is found in the volumes of basement
TABLE 4.
Comparison of Results Obtained by Two Different Laboratories, Using the Same Slides from (Relative Volumes Are Expressed in % Total Testicular Volume)
Iowa
Structure
Leydig cells Basement membrane and tunica propria
Spermatogonia Spermatocytes Spennatids and spermatozoa Total germ oells (iocluding abnormals) Sertoli oells Lumen Space
92 Biopsy Specimens
Washington Mean difference between devlation.o af individual cases from their S.D. respective means
Mean relative volume
Rtmge
S.D.
Mean -relative volume
Range
3.6
0.1 10.8
1.96
3.0
0.3- 8.4
1.91
0.93
13.0 7.3 11.8 7.3
5.3-25.8 3.0-12.4 1.1-18.6 0.0-14.9
3.68 1.84 3.28 3.21
9.6 7.1 11.4 6.1
5.3-17.1 2.0-11.0 1.0-20.7 0.0-11.4
2.28 1.80 3.39 2.65
2.42 1.32 1.56 1.45
27.3 18.0 7.1 5.2
6.6-40.7 11.8-33.6 1.3-19.5 1.0-12.0
6.48 4.32 3.13 2.50
25.4 19.8 8.4 4.4
5.5-37.2 8.4-33.9 0.6-22.6 2.3-10.1
6.34 4.86 4.05 1.64
2.64 2.92 1.87 2.12
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membrane and tunica propria. The limits of these structures were arbitrarily defined (see paper I) and it is obvious that the laboratories did not make the same identifications, probably because the definitions were not sufficiently rigid. On the other hand, items for which there was complete agreement on the criteria of identification, such as, for instance, spermatogonia · or spermatocytes, show very small differences. The last column in Table 4 conveys some information concerning the differences in individual cases. It may be seen that the differences when adjusted to the respective means are usually far smaller than the standard deviations. This indicates fairly good agreement. There were only 2 cases in which the results of the two laboratories differed sufficiently to lead to essentially different diagnoses. The most important result of the comparison appears to be the demonstration that excellent agreement can be reached on the most significant diagnostic items, i.e., the germ cells. DISCUSSION
In the preceding pages data have been presented concerning volume proportions of testicular tissue elements in various pathologic and nonpathologic conditions. In evaluating the data we made strenuous efforts to sift from them some information leading to the establishment of certain patterns of spermatogenic defects. It may be assumed that the agents which caused deviations from the norm in our specimens were varied in quality and quantity. In fact, this is almost assured by the random selection of patients and the lack of detailed information about many of them. The question arises whether the germinal epithelium and its supportive elements react in definite, classifiable ways to pathologic stimuli and whether these responses, if present, can be recognized by means .of data pertaining to volume proportions. Reactions af Germ Cells
Theoretically one might postulate the occurrence of any of the following reactions of the germ cells to noxious agents: 1. The spermatogonia die or become unable to divide. This leads to a complete disappearance of germ cells except, perhaps, for a small number of spermatogonia. Atrophy of this type was excluded from our series of cases and will not be considered here.
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2. The spermatogonia are kept from restoring their normal numbers, their mitoses are hindered or slowed, and a diminished number of spermatocytes is formed. These may develop normally but the output in spermatozoa is low. This condition might be called "simple or proportional hypoplasia." In it the total germ cell volume and the volumes of all gertn cell generations should be low, but the proportions of the generations should be approximately normal. Among 96 biopsies which showed any deficiencies in germ cell volumes, there were 10 of this type. Seven occurred in the nitrofuran series ( 2 before, 5 after treatment). The average germ cell volume of the 10 cases was only slightly below normal range ( 23.8 per cent, with 3 cases 25.2 per cent; lowest normal value 25.5 per cent). It is probable, therefore, that some of these testes were not abnormal at all, but represent extensions of the "normal" range which was established by only 30 specimens (paper I). The condition of "simple hypoplasia" appears to be rare and some doubt exists whether it can ever be a permanent state. Certain cases of pituitary tumor or severe irradiation hypoplasia may present the picture of simple and permanent decrease in all germ cell generations but no quantitative analyses of the spermatogenic pattern in these conditions are. available at present. 3. The spermatogonia may be unaffected by the injurious agent, but in the subsequent differentiation and maturation divisions of the spermatocytes or in spermiogenesis an abortion of cellular development may occur. Our results indicate that this takes place most frequently at the stage of the spermatid. In 36 testes ( 38 per cent of all deficient specimens) the volume of spermatids and spermatozoa was subnormal with no appreciable decrease in the volume of either spermatocytes or spermatogonia. In addition, there were 18 cases in which all germ cell generations were diminished with a disproportionately large decrease in spermatids and spermatozoa. Indications of arrest at the level of the spermatids were, therefore, present in more than half of the abnormal testes. Arrest at the spermatocyte stage was infrequent. There were only 6 cases (in the oligospermic group and the miscellaneous cases) which showed subnormal volumes of spermatocytes and later stages with normal or high normal spermatogonial volumes. In an additional 5 cases all generations had volumes below normal range but spermatocytes, spermatids, and spermatozoa were relatively much more decreased than spermatogonia. Thus 11 specimens presented varying degrees of what Engle has called
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"spermatogenic" arrest. We would prefer the terms "spermatocytic" or "early spermatogenic" arrest in order to distinguish this condition from the very common "spermatid" or "late spermatogenic" arrest. Spermatocytic arrest is a failure of most spermatocytes to pass normally through the first maturation division. The volume of spermatocytes should be considerably diminished but an appreciable volume should still be recorded for the primary spermatocytes. In fact, our cases of this type had an average spermatocytic volume of 5.5 per cent (normal mean 14.4 per cent). Abnormal cells had a mean volume of 1.1 per cent (normal mean 0.39 per cent), an increase which is probably significant. No attempt was made at this time to determine the exact stages at which spermatogenesis in the human may become abortive. It may be pointed out, however, that in the rat and other animals the first maturation division and the early differentiation of the spermatozoon are two periods in the life of the germ cell during which degeneration and cell death often occur even in the normal testis. 7 It would not be surprising if these two stages were particularly involved in "early" and "late" spermatogenic arrest. 4. Among the theoretical possibilities of pathologic reactions of the germinal epithelium is a change in timing. It is conceivable that all processes in an otherwise normal pattern of spermatogenesis might become so slowed that the output of spermatozoa is greatly decreased even though volume proportions appear undisturbed. If there were any evidence, even from animal experiments, that this ever happens, it might provide a partial explanation for the lack of correlation between the quantitative histology of the testis and the sperm counts. Unfortunately, there is to our knowledge no evidence at all and the concept remains purely speculative. Other Conclusions The data obtained by Chalkley's method do not appear at the present time to permit a classification of spermatogenic defects beyond the categories of "late arrest" (more than 50 per cent of our cases), "early arrest" (about 10 per cent), and "simple hypoplasia" (certainly below 10 per cent, perhaps below 5 per cent). Special attention was paid to the correlation between Leydig cell volume and spermatogenic deficiency. The mean volume of Leydig cells was significantly decreased in all groups of cases with the somewhat surprising exception of the oligospermic and uormospermic infertility series which
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showed a normal average. However, in only 16 cases was the volume of Leydig cells actually outside the normal range; iu 10 cases it was below, iu 6 above the norm. In individual cases there appeared to be no consistent relationship between the volume of Leydig cells and the state of the seminiferous tubules. In one specimen with nearly normal spermatogenesis the Leydig cells occupied less than 0.1 per cent of the biopsy. Thus it appears that the volume of Leydig cells is usually not a diagnostic feature, particularly when the large range in the normal testis is considered ( 0.6-5.8 per cent). The volumes of the layers which envelop the seminiferous tubules (basement membrane and, tunica propria) were significantly iucreased in the cases of infertility with patent ducts and, even more so, in the miscellaneous group, but in many cases with spermatogenic deficiencies they were normal. Of 96 cases, 27 showed an increase above the normal maximum of 12.5 per cent. In individual cases the correlation between the degree of abnormality of the germ cells and the thickness of basement membrane and tunica was not close, but the group as a whole manifested severe damage as seen, for instance, in the low average volume of only 2.2 per cent for spermatids and spermatozoa (normal range 5.0-12.2 per cent). There were 3 cases of normal spermatogenesis with volumes of basement. membrane and tunica slightly above the normal range. These probably represent extensions of the "norm." An increased volume of interstitial space exclusive of Leydig cells, basement membrane, and tunica propria was recorded in 112 biopsies ( 62 per cent of all specimens). However, this finding was almost as frequent iu the normal as in the abnormal testes. We feel that it represents, for the most part, the results of artifacts which are typical for biopsy specimens. The handling of the tissues at operation usually results either in a decrease or, more often, in an increase of the interstitial spaces which become filled with fluid, blood, or even germ cells (paper I). It is, therefore, difficult if not impossible to use the volume of interstitial space as an iudication of fibrosis. On the other hand, there are cases-and several were found iu our materialill which the fibrous tissue in the interstitium and particularly around the vessels is greatly increased, although there is no appreciable increase in the tunica propria. This type of fibrosis appears to be different from that implied by a large volume of basement membrane and tunica propria. Our data can, therefore, not be used to. quantitate "general fibrosis" within the testis.
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In the present paper we have attempted a quantitative analysis of testicular biopsy specimens, and from our results we draw the conclusion that Chalkley's method is a useful tool in such attempts. Within limits which we have tried to define, the method gives detailed information through relatively simple procedures. We have applied it, however, in less than optimal form because no attempt was made to obtain absolute values concerning tissue volumes. In future investigations it should be possible to repair this omission by taking a few simple, external measurements of the gonad at the time of biopsy and estimating from these the absolute volume of the testes. 7 In this way the Chalkley method could be made to yield absolute data concerning the mass of germinal epithelium, Leydig cells, etc. It is very clear to us that all conclusions drawn from our data are subject to the limitations of our normal standards of comparison. The original normal series was small but carefully selected for "quality" of the testicular tissues. Experience with the present series, which contains six times as many specimens, makes us feel that the standard of comparison is probably too "good"; that a random series of healthy men would show lower means for germ cell volumes, and probably for Leydig cells. Quite certainly the ranges of "normalcy" are greater by 10-20 per cent than those given in paper I, and this must be taken !nto consideration in any future work on this subject.
SUMMARY Chalkley's method for determining the relative volume of tissue components in histologic sections was applied to 180 human testicular biopsies from 125 men with various conditions, among them infertility with azospermia, oligospermia, and normospermia. Results for various categories of cases have been presented and briefly discussed. The data obtained lead to the following classification of the cases with deficient spermatogenesis studied in this series: ( 1) "Late spermatogenic or spermatid arrest." This occurred in more than half of the specimens. It was identified by a disproportional decrease in the volume of spermatids and spermatozoa. ( 2) "Early spermatogenic or spermatocytic arrest" ( 10 per cent of the cases). This was recognized by a disproportional decrease in volume of spermatocytes, spermatids, and spermatozoa. ( 3) "Simple or proportional hypoplasia" (less than 10 per cent of the cases). This was characterized by a proportional decrease of all generations of germ cells. Volumes of Leydig cells were in the average decreased but in individual
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cases the correlation between Leydig cell volume and spermatogenic deficiency was very poor. Volumes of basement membrane and tunica propria were in the average increased in most categories of cases. Such an increase was usually, but not always, associated with subnormal spermatogenesis. It was pointed out that an increase in the bulk of the immediate envelopes of the seminiferous tubules is an indication of only one type of fibrosis occurring in the testis. Sperm counts from 1-105 millionjcc. correlated poorly with the degrees of deficiency of the germinal epithelium. All cases with counts from "very few" to 0.9 million/cc. had greatly diminished germ cell volumes. • The usefuloess and limitations of Chalkley's method in evaluating testicular biopsies have been discussed. REFERENCES 1. CHALKLEY, H. W. ]. Nat. Cancer Inst. 4:47, 1943. 2. CluRNY, C. W. In Diagnosis in Sterility. Proceedings of the Conference on Problems of Human Sterility, 1946. 3. ENGLE, E. T. Ann. New York Acad. Sc. 55:703, 1952. 4. NELSON, W. 0. lo Studies on Testis, and Ovary, Eggs and Sperm. Proceedings of the Conference Committee on Human Reproduction, 1952. 5. NELSON, W. 0., and STEINBERGER, E. Anat. Rec. 112:367, 1952. 6. NELseN, W. 0., and BuNGE, R. G. ]. U
8
MAY-JUNE,
1957
NUMBER
3
Quantitative Investigations on Human Testicular Biopsies II. Infertility and Other Conditions
Edward C. Roosen-Runge, M.D., Eve Marberger, M.D., and Warren 0. Nelson, Ph.D.*
IN
THE PRECEDING PAPER9 (hereafter referred to as paper I) 26 normal human testes were analyzed with respect to the volume proportions of their histologic elements, and some standards for the evaluation of testicular biopsies were established. Chalkley's method was used, and described and discussed in detail. As a result of this study it was decided to investigate the volume proportions of testicular components in various abnormal conditions which had never previously been explored quantitatively. Two major objectives were sought: ( 1) to define certain abnormal states of sperma-
From the Departments of Anatomy, University of Washington School of Medicine, Seattle, Wash., and State University of Iowa, College of Medicine, Iowa City, Iowa. Aided by grants from the National Science Foundation (G-516) and the U.S. Public Health Service (RG-1778). Thanks are due to Mrs. J. A. Gromons for able technical assistance. • Present address: Rockefeller Institute, New York, N. Y. 203
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to genesis in quantitative terms, and ( 2) to ascertain· the degree of applicability of the method to clinical investigations. MATERIAL AND METHOD
A total of 180 testicular biopsies from 125 men were analyzed. Bilateral biopsies were available in 42 cases. The specimens were wedges of tissue obtained by experienced operators and preserved immediately in Bonin's or Stieve's solution. Sections were stained with various trichrome stains and in some cases with Gomori's Aldehyde-Fuchsin method. The cases fell into several categories, which will be treated separately below. The classification of the material is indicated in the first 3 columns of Table l. Clinical diagnosis and evaluation were minor factors in the selection of cases for this study. The main objective was to obtain a fairly large number of well-preserved biopsy specimens from a variety of cases. Only specimens were used which showed a fair number of germ cells. Cases with germinal cell aplasia, complete spermatocytic arrest, or severe peritubular fibrosis were not studied. The diagnosis of "infertility" was made in men who had been unable to effect a conception during a period of at least 1 year-in most instances it was several years. In the large majority of cases, the wives had been examined and pronounced potentially fertile. The average ages of the first 4 groups of cases of Table 1 were almost identical. They ranged from a mean of 27.8 years for the nitrofuran cases, to 30.0 years for cases of infertility with oligospermia or normospermia. In the miscellaneous cases the average age was significantly greater: 43 years. The average age of the cases of infertility without sperm counts could not be determined. Chalkley's method will not be described here, as extensive descriptions of the procedure have recently been published.'· 10 It appears necessary, however, to explain once again some of the terms used in this and the previous paper. The term "space" is used for the structures in the seminiferous tubules which could not be identified, regardless of whether or not they appeared to be parts of cells or artifacts. The term "lumen" does not seem to need a specific explanation, but it should be kept in mind that in biopsy specimens the lumen of the tubules is often filled with cells in addition to a fine coagulum. In these cases the cells were recorded in the same way as
TABLE 1.
Classification of Cases and Means of Volume Proportions of Testicular Components Compared with Normal Value~ Testicular components
Condition
No. patients
Normal (standard error of the mean in p"a.rentheses) 26
No. biopsies
Basement membrane and tunica
propria
Leydig cells
Spermatids and Spermato- Spermato- spemwcytes tozoa gonia
14.4 (0.47)
9.1 (0.36)
SertoU ceUs
17.4 (0.38)
Lumen
Total germ cells (including abnormals)
10.6 (0.59), 9.8 for 24
32.0 (0.68)
30
9.1 (0.33)
3.1 (0.23)
7.8 (0.20)
15
23
9.3
2.2
7.4
13.3
7.3
17.8
8.5
28.6
56
80
10.4
3.0
7.5
10.6
4.6
20.7
8.1
23.7
14 14 9 17
21 21 12 23
9.4 8.9 9.9 11.5
2.1 2.7 1.5 2.3
6.9 7.1 7.9 6.5
12.0 13.0 10.4 12.3
7.2 7.2 5.2 3.2
18.8 20.2 19.7 20.3
6.1 8.1 8.4 8.9
26.8 27.8 24.5 23.3
biopsies
Infertility with azoospermia Infertility with oligo- or nonnospermia Nitrofuran treatment Before Mter Infertility, no sperm count Miscellaneousa cases
a Four patients with schizophrenia, 2 with Addison's disease, 2 with "delayed puberty," 2 with hydrocele, 2 over 70 years old, 1 each with myxedema, sickle cell anemia, gyr.ecomastia, other testicle cryptorchid and one without diagnosis.
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those in their natural positions; the coagulate was recorded as lumen. Additional definitions of terms may be found in paper I. INFERTILITY WITH AZOOSPERMIA
It can be assumed that all cases of this group had some obstruction of the seminal passages, as the seminiferous tubules appeared to produce some normal, mature spermatozoa in all of them. The mean volumes of this group are recorded in Table 1. In most instances, differences from the normal standards are slight. The most significant deviation occurs in the volume of the total germ cells, which is 28.6 per cent, as compared with the normal mean of 32 per cent. More than half of this difference is accounted for by a comparatively low volume of spermatids and spermatozoa. The much smaller difference in the volume of the spermatogonia is statistically not significant. In general, the azoospermic patients show fewer deviations from the normal than any other group investigated. A detailed analysis of the 15 cases ( 23 testes) showed that 8 ( 13 testes) were within normal range in all values. In 2 cases 1 testis was normal, the other showed a decrease of the total germ cell volume to just below the normal range ("hypoplasia''). In 2 other cases both testes showed a decrease in total germ cell volume. In these 4 testes the volume of spermatids and spermatozoa was dispropmtionately low, probably indicating some "arrest" of spermatogenesis at the spermatid stage. In another 2 cases ( 2 testes) severe decreases in total germ cell volume were recorded ( 14.6 and 20.7 per cent) and, in addition, the volume of spermatids and spermatozoa was below 50 per cent of the lowest value in the normal series. The data confirm quantitatively what has been known for some time, that spermatogenesis is not appreciably affected in men with obstruction of the ducts. Nelson' has presented a series of 21 cases with blocked or absent efferent ducts. In 8 cases the spermatogenic pattern was inferior, but this did not appear to be related to the defect in the seminal passages. In the present series, one third of the cases showed inferior spermatogenesis and in 2 cases a severe degree of deficiency was present. This proportion appears to be no greater than in a randomly selected series of men who have no obvious defects of reproduction (see nitrofuran cases below). On the other hand, it should be emphasized that the control series (paper I) was not randomly selected but carefully chosen for "normality" of the testicular tissues.
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The view is often expressed in pathologic practice that widened lumina of seminiferous tubules are frequently found in patients with obstruction of the ducts. Our data do not support this view. Only one specimen showed a somewhat increased relative volume of the lumen and the average value for lumen is comparatively low. INFERTILITY WITH OLIGOSPERMIA AND NORMOSPERMIA In the average, the cases in this group show a more inferior quantitative picture than the preceding category. Basement membrane and tunica propria are increased in volume, the value for total germ cells is low, the TABLE 2.
Correlation of Sperm Counts and Relative Volume of Total Germ Cells in the Testis in 80 Biopsy Specimens fro~:n Patients with ''Infertility''
Counts
From "very few" to . 0.9mil./cc. 1-10 mil.jcc. 11-50 mil./cc. 51-105 mil./cc.
No. specimens
Relative volume of total germ cells ln %af testis volume
17 26 26 11
16 26 26 26
volume of spermatids and spermatozoa is greatly disproportionately de" creased, and the Sertoli cells show higher than normal values. Both testes were found to be within normal limits in 7 cases. In 2 cases one testis was normal, the other abnormal to a slight extent. Of 80 testes, 56 were definitely abnormal. Of these, 40 had a relative volum~ of spermatids and spermatozoa below 5 per cent (the lowest limit of "normal"). In 21 cases only the spermatids and spermatozoa were decreased, indicating arrest at the early spermatid level, in 6 cases the spermatogonia alone appeared to be of normal volume (arrest at early spermatocyte level), and in 9 cases the volumes of all germ cell generations were proportionately decreased (proportional hypoplasia). An increase in basement membrant: and tunica propria was recorded in 17 cases; the highest value was 18.1 per cent. Sertoli cell volume was increased to an average of 20.7.per cent in the 40 specimen,s and was associated with subnormal volumes of spermatids and spermatozoa in 70 per cent of these cases. Each of the 56 patients in this group had at least orie sperm count, the
TABLE 3.
Total sperm count Case no. (millions) la. R
L lb. R
L 2a. L 2h.R Sa. R L Sb.R L 4a. R L 4b.R L 5a. R 5b.R L 6a. L 6b.R L 7a. R L 7b.R L Sa. R
30 00
No count
..
40 No count
29 00
No count
..
46
..
No count
..
547• 503•
..
No count
No count 00
480
..
No count 00
223
Relative Volumes of Testicular Components in Cases (a) before and (b) after Treatment with Nitrofuran
Basement membrane Spermato~
Spermat~
propria
Leydig cells
gonia
cytes
8.6 9.9 6.4 9.6 7.4 7.1 11.7 9.9 11.2 7.1 7.7 10.7 7.9 8.3 7.0 10.6 8.7 11.0 5.6 8.1 8.9 8.1 9.8 7.6
0.4 1.4 0.7 1.4 2.3 3.0 1.0 2.3 1.4 1.3 1.4 1.3 4.4 1.1 2.6 2.7 3.9 3.3 4.8 4.0 1.4 4.1 2.9 3.3 4.0
5.7 7.7 8.5 8.3 4.0 6.6 4.7 7.1 6.0 8.1 6.3 7.4 5.0 5.9 6.9 7.0 7.1 5.6 5.4 6.7 7.7 7.5 10.3 9.6 7.7
and tunica
9.4
15.0 15.0 13.3 20.7 5.7 9.6 11.6 12.1 10.7 13.4 14.4 13.0 13.4 14.7 10.1 9.4 12.1 11.1 11.7 14.6 12.2 11.9 15.3 15.0 10.6
Spermatid$ Abnormal and germ spermatozoa cells
6.7 8.7 7.0 7.6 2.3 6.4 7.6 8.9 6.3 6.9 11.0 9.7 10.4 5.3 6.4 7.7 6.9 5.0 7.0 8.4 8.7 7.1 8.6 8.0 7.1
1.7 1.6 0.7 0.6 0.3 1.3 0.4 0.3 0.1 0.4 0.7 0.7 0.3 0.9 0.4 1.1 1.0 1.0 0.6 0.1 1.1 0.6 0.6 0.4 1.0
Total germ Sertoli cells
Lumen
cells
18.9 15.8 16.9 18.0 16.7 21.8 20.7 15.3 20.9 22.7 17.4 21.7 23.6 20.7 23.1 18.1 25.0 19.3 17.4 24.2 17.2 15.3 18.8 22.3 17.1
2.4 9.6 8.2 6.6 7.0 11.6 10.1 10.4 6.1 8.9 9.1 5.2 11.9 5.0 4.6 6.3 4.3 3.1 11.4 8.1 9.7 11.8 8.3 11.4 6.5
29.1 33.0 29.5 37.2 12.3 23.9 24.3 28.4 23.1 28.8 32.4 30.8 29.1 26.8 23.8 25.2 27.1 22.7 24.7 29.8 29.7 27.1 34.8 33.0 26.4
L 8b. R L 9a. R L 9b. R lOa. R IOb;L lla. R lib. L 12a. L 12b. R 13a. R L l3b.L 14a. L l4b.R a
.. .. 172 ..
No count
No count No count No count
340• 100 730 240 No count
..
No count
853 No count
9.7 8.1 8.0 10.3 15.9 10.0 8.9 9.7 10.0 8.1 8.0 ll.3 9.1 8.3 13.7 7.7 10.7
Count done 1--S weeks after biopsy.
1.0 2.3 l.l 1.3 1.0 2.1 2.3 3.7 4.6 3.7 2.0 3.7 l.l 2.4 1.9 2.6 4.6
6.0 8.2 6.1 9.4 7.7 7.4 6.3 5.3 7.1 7.6 9.9 5.7 6.4 7.0 8.1 6.0 6.6
10.4 13.3 15.3 14.9 12.1" 15.6 12.4 10.7 10.9 10.2 12.7 10.2 14.3 ll.3 12.9 11.0 10.3
6.9 9.8 8.9 11.0 4.7 7.9 8.0 5.7 4.0 7.7 9.4 5.1 5.6 4.1 3.1 7.4 7.3
1.6 0.7 1.0 0.6 0.7 0.6 0.4 0.1 0.4 0.1 0.3 0.7 0.9 0.6 0.1 0.4 0.1
2l.l 18.2 24.1 16.8 16.9 16.7 22.0 18.4 28.7 24.6 19.0 17.1 15.3 14.7 16.7 21.4 18.4
5.9 9.0 8.3 6.6 3.4 10.0 9.2 7.0 8.4 9.3 14.6 3.9 9.7 9.2 7.6 8.1 6.7
24.9 32.0 31.3 35.9 25.2 31.2 27.1 21.8 22.4 25.6 32.3 21.7 27.2 23.0 24.2 25.7 24.3
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majority had two or more. The counts were done in various laboratories and were not rigidly comparable nor reliable. Therefore, only a rough estimate of the correlation between volume measurements and sperm counts is attempted in Table 2. It appears that there is no appreciable correlation with the sperm counts above 1 million fcc. of semen but that very low counts are usually associated with a severe decrease in germ cells. Analysis of the individual cases confirmed this impression and showed that all germ cell volumes below 19 per cent had counts of 25 millionjcc. or less. Sixteen specimens ( 20 per cent) fell into this category. While this definite positive correlation is of real significance, the lack of correlation for counts ranging from 1 to 105 million/cc. is probably not significant in view of the technics by which some of the counts were obtained. In addition, the counts were done in ahnost all cases either at the time of biopsy or weeks to months before biopsy. CASES BEFORE AND AFTER TREATMENT WITH NITROFURAN The specimens in this group were obtained in the course of an investigation by Nelson and Bunge' on the effect of therapeutic doses ( 5-10 mg./kg.) of Furadantin on human spermatogenesis. It had been shown earlier' that the various nitrofurans have the capacity of arresting spermatogenesis at the spermatocyte stage in the rat. In the present paper only the quantitative analyses of human testicular biopsies will be reported. At least two biopsies, taken at an interval of about 2 weeks, were available in every case, and in 5 individuals two successive biopsies were taken from each testis. The data are shown in detail in Table 3, which may serve as an example of the type of information which forms the basis for all results presented in this paper. The mean values of the group are recorded in Table 1. No significant quantitative differences in volume proportions were observed after treatment with nitrofuran. This essentially confirms Nelson and Bunge's impressions. There are in this series 15 testes with deficient spermatogenesis, and the average relative volumes are slightly more abnormal than in the group with azoospermia. (All subnormal biopsies came from inmates of a prison who volunteered for the experiments.) Once again no correlation could be demonstrated between sperm counts and defects in spermatogenesis, although the counts in this group were relatively well controlled and their timing with respect to the biopsies was favorable for the establishment of correlations. In 5 cases high sperm counts
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were associated with subnormal germ cell volumes. In case 5, for example, the two biopsies of the right testis showed subnormal volumes of all germ cell generations and the total germ cell volume of the left testis (after treatment) was in the lowest normal range. Sperm counts, however, which were done 5 and 2 days before and 5, 9, 16, and 60 days after the first biopsy all showed high normal values. Results were similar in cases 11, 12, and 14, and in 1 case of the normospermic infertility group. In every one of these 5 cases the quantitative deficiency consisted of a proportional decrease in all germ cell generations and no "arrest" was observed. The findings might be explained by assuming a large total volume of the testes in these cases. Exceptionally large testes might well produce a high sperm count even if the average productivity of the seminiferous epithelium is low. How similar are biopsies from the same individual or from the same testis after 2 weeks? Our results appear to indicate- that they are in general quite similar, particularly if the same testis is analyzed. Usually the two testes of the same individual are also similar, but exceptions are seen, for instance, in cases 2 and 12. Among the 42 cases in which simultaneous bilateral biopsies were done, 10 differed to such a degree that one testis was definitely abnormal while the other one was normal. Charny has emphasized "startling similarity" of biopsy specimens removed before and after treatment from the same testis. From our observations we can state that the similarity is usually more impressive qualitatively than quantitatively. MISCELLANEOUS CASES Diagnoses and volume proportions of these cases are summarized in Table 1 and footnote. Most patients of this group suffered from definite diseases and showed in general far more deficient spermatogenesis than the patients with complaints of infertility. It must, however, be kept in mindthat the average age of this group was much higher than that of any of the other categories. · Three out of 4 cases with schizophrenia showed relatively low volumes of spermatids and spermatozoa (indication of "arrest"),'but in only 1 case was the total volume of germ cells below normal limits. In general, the biopsies from schizophrenic patients were among the most normal of the miscellaneous series.
One of the 2 cases with Addison's disease had a quantitatively normal biopsy (sperm counts 110-140 millionjcc.). The other (no sperm counts)
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had a greatly decreased germ cell volume with a very low volume of spermatids and spermatozoa ( 1.9 per cent). The Leydig cell volume was entirely normal in both cases. Subnormal germ cell volumes were found in the biopsy specimens from 2 cases with the diagnosis of "delayed puberty." In one case neither spermatids nor spermatozoa were present; the spermatogonial volume was 4.9 per cent, the spcrmatocytic volume 1.1 per cent. The other case presented a picture of less severe arrest with 2.9 per cent spermatids and spermatozoa. In 2 cases with hydrocele the basement membrane and tunica propria were greatly increased and the volume proportions of the germ cells indicated arrest, with normal spermatogonial volume. Biopsies from 2 men, 70 and 74 years old, showed a general decrease in all generations of germ cells, with the most severe decline in the spermatids and spermatozoa ( 0.7 and 3.2 per cent) and slight increases in the basement membrane and tunica propria. A case of myxedema also had a general and severe decrease in germ cells and an increase in basement membrane and tunica, but this was one of the rare cases in which spermatids and spermatozoa appeared in the normal proportion although the total volnme of germ cells was only 10 per cent. ' A patient with sickle cell anemia had a quantitatively normal biopsy specimen. An individual with gynecomastia ( 47 years old) showed low genn cell volumes in both testes ( 18.6 and 17.9 per cent) with disproportional . decrease in spermatids and spermatozoa and an increase in basement membrane and tunica propria. In the scrotal testis of an individual ( 19 years old) who had one cryptorchid testis, severe disproportional decrease of spermatids and spermatozoa was recorded but .no other abnormalities were found. COMPARISON OF RESULTS IN TWO DIFFERENT LABORATORIES The results presented in the preceding sections of this paper are based entirely on counts made in one laboratory (Washington). Table 4 permits a comparison of data from two laboratories who worked independently with Chalkley's method on the identical preparations from 92 randomly selected cases. The comparison adds to our information concerning the reliability of the method when applied to testicular biopsies. It also con£rms the crucial importance of the criteria of identification. The greatest difference in the data obtained by the two laboratories is found in the volumes of basement
TABLE 4.
Comparison of Results Obtained by Two Different Laboratories, Using the Same Slides from (Relative Volumes Are Expressed in % Total Testicular Volume)
Iowa
Structure
Leydig cells Basement membrane and tunica propria
Spermatogonia Spermatocytes Spennatids and spermatozoa Total germ oells (iocluding abnormals) Sertoli oells Lumen Space
92 Biopsy Specimens
Washington Mean difference between devlation.o af individual cases from their S.D. respective means
Mean relative volume
Rtmge
S.D.
Mean -relative volume
Range
3.6
0.1 10.8
1.96
3.0
0.3- 8.4
1.91
0.93
13.0 7.3 11.8 7.3
5.3-25.8 3.0-12.4 1.1-18.6 0.0-14.9
3.68 1.84 3.28 3.21
9.6 7.1 11.4 6.1
5.3-17.1 2.0-11.0 1.0-20.7 0.0-11.4
2.28 1.80 3.39 2.65
2.42 1.32 1.56 1.45
27.3 18.0 7.1 5.2
6.6-40.7 11.8-33.6 1.3-19.5 1.0-12.0
6.48 4.32 3.13 2.50
25.4 19.8 8.4 4.4
5.5-37.2 8.4-33.9 0.6-22.6 2.3-10.1
6.34 4.86 4.05 1.64
2.64 2.92 1.87 2.12
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membrane and tunica propria. The limits of these structures were arbitrarily defined (see paper I) and it is obvious that the laboratories did not make the same identifications, probably because the definitions were not sufficiently rigid. On the other hand, items for which there was complete agreement on the criteria of identification, such as, for instance, spermatogonia · or spermatocytes, show very small differences. The last column in Table 4 conveys some information concerning the differences in individual cases. It may be seen that the differences when adjusted to the respective means are usually far smaller than the standard deviations. This indicates fairly good agreement. There were only 2 cases in which the results of the two laboratories differed sufficiently to lead to essentially different diagnoses. The most important result of the comparison appears to be the demonstration that excellent agreement can be reached on the most significant diagnostic items, i.e., the germ cells. DISCUSSION
In the preceding pages data have been presented concerning volume proportions of testicular tissue elements in various pathologic and nonpathologic conditions. In evaluating the data we made strenuous efforts to sift from them some information leading to the establishment of certain patterns of spermatogenic defects. It may be assumed that the agents which caused deviations from the norm in our specimens were varied in quality and quantity. In fact, this is almost assured by the random selection of patients and the lack of detailed information about many of them. The question arises whether the germinal epithelium and its supportive elements react in definite, classifiable ways to pathologic stimuli and whether these responses, if present, can be recognized by means .of data pertaining to volume proportions. Reactions af Germ Cells
Theoretically one might postulate the occurrence of any of the following reactions of the germ cells to noxious agents: 1. The spermatogonia die or become unable to divide. This leads to a complete disappearance of germ cells except, perhaps, for a small number of spermatogonia. Atrophy of this type was excluded from our series of cases and will not be considered here.
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TESTICULAR BIOPSIES, II
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2. The spermatogonia are kept from restoring their normal numbers, their mitoses are hindered or slowed, and a diminished number of spermatocytes is formed. These may develop normally but the output in spermatozoa is low. This condition might be called "simple or proportional hypoplasia." In it the total germ cell volume and the volumes of all gertn cell generations should be low, but the proportions of the generations should be approximately normal. Among 96 biopsies which showed any deficiencies in germ cell volumes, there were 10 of this type. Seven occurred in the nitrofuran series ( 2 before, 5 after treatment). The average germ cell volume of the 10 cases was only slightly below normal range ( 23.8 per cent, with 3 cases 25.2 per cent; lowest normal value 25.5 per cent). It is probable, therefore, that some of these testes were not abnormal at all, but represent extensions of the "normal" range which was established by only 30 specimens (paper I). The condition of "simple hypoplasia" appears to be rare and some doubt exists whether it can ever be a permanent state. Certain cases of pituitary tumor or severe irradiation hypoplasia may present the picture of simple and permanent decrease in all germ cell generations but no quantitative analyses of the spermatogenic pattern in these conditions are. available at present. 3. The spermatogonia may be unaffected by the injurious agent, but in the subsequent differentiation and maturation divisions of the spermatocytes or in spermiogenesis an abortion of cellular development may occur. Our results indicate that this takes place most frequently at the stage of the spermatid. In 36 testes ( 38 per cent of all deficient specimens) the volume of spermatids and spermatozoa was subnormal with no appreciable decrease in the volume of either spermatocytes or spermatogonia. In addition, there were 18 cases in which all germ cell generations were diminished with a disproportionately large decrease in spermatids and spermatozoa. Indications of arrest at the level of the spermatids were, therefore, present in more than half of the abnormal testes. Arrest at the spermatocyte stage was infrequent. There were only 6 cases (in the oligospermic group and the miscellaneous cases) which showed subnormal volumes of spermatocytes and later stages with normal or high normal spermatogonial volumes. In an additional 5 cases all generations had volumes below normal range but spermatocytes, spermatids, and spermatozoa were relatively much more decreased than spermatogonia. Thus 11 specimens presented varying degrees of what Engle has called
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"spermatogenic" arrest. We would prefer the terms "spermatocytic" or "early spermatogenic" arrest in order to distinguish this condition from the very common "spermatid" or "late spermatogenic" arrest. Spermatocytic arrest is a failure of most spermatocytes to pass normally through the first maturation division. The volume of spermatocytes should be considerably diminished but an appreciable volume should still be recorded for the primary spermatocytes. In fact, our cases of this type had an average spermatocytic volume of 5.5 per cent (normal mean 14.4 per cent). Abnormal cells had a mean volume of 1.1 per cent (normal mean 0.39 per cent), an increase which is probably significant. No attempt was made at this time to determine the exact stages at which spermatogenesis in the human may become abortive. It may be pointed out, however, that in the rat and other animals the first maturation division and the early differentiation of the spermatozoon are two periods in the life of the germ cell during which degeneration and cell death often occur even in the normal testis. 7 It would not be surprising if these two stages were particularly involved in "early" and "late" spermatogenic arrest. 4. Among the theoretical possibilities of pathologic reactions of the germinal epithelium is a change in timing. It is conceivable that all processes in an otherwise normal pattern of spermatogenesis might become so slowed that the output of spermatozoa is greatly decreased even though volume proportions appear undisturbed. If there were any evidence, even from animal experiments, that this ever happens, it might provide a partial explanation for the lack of correlation between the quantitative histology of the testis and the sperm counts. Unfortunately, there is to our knowledge no evidence at all and the concept remains purely speculative. Other Conclusions The data obtained by Chalkley's method do not appear at the present time to permit a classification of spermatogenic defects beyond the categories of "late arrest" (more than 50 per cent of our cases), "early arrest" (about 10 per cent), and "simple hypoplasia" (certainly below 10 per cent, perhaps below 5 per cent). Special attention was paid to the correlation between Leydig cell volume and spermatogenic deficiency. The mean volume of Leydig cells was significantly decreased in all groups of cases with the somewhat surprising exception of the oligospermic and uormospermic infertility series which
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showed a normal average. However, in only 16 cases was the volume of Leydig cells actually outside the normal range; iu 10 cases it was below, iu 6 above the norm. In individual cases there appeared to be no consistent relationship between the volume of Leydig cells and the state of the seminiferous tubules. In one specimen with nearly normal spermatogenesis the Leydig cells occupied less than 0.1 per cent of the biopsy. Thus it appears that the volume of Leydig cells is usually not a diagnostic feature, particularly when the large range in the normal testis is considered ( 0.6-5.8 per cent). The volumes of the layers which envelop the seminiferous tubules (basement membrane and, tunica propria) were significantly iucreased in the cases of infertility with patent ducts and, even more so, in the miscellaneous group, but in many cases with spermatogenic deficiencies they were normal. Of 96 cases, 27 showed an increase above the normal maximum of 12.5 per cent. In individual cases the correlation between the degree of abnormality of the germ cells and the thickness of basement membrane and tunica was not close, but the group as a whole manifested severe damage as seen, for instance, in the low average volume of only 2.2 per cent for spermatids and spermatozoa (normal range 5.0-12.2 per cent). There were 3 cases of normal spermatogenesis with volumes of basement. membrane and tunica slightly above the normal range. These probably represent extensions of the "norm." An increased volume of interstitial space exclusive of Leydig cells, basement membrane, and tunica propria was recorded in 112 biopsies ( 62 per cent of all specimens). However, this finding was almost as frequent iu the normal as in the abnormal testes. We feel that it represents, for the most part, the results of artifacts which are typical for biopsy specimens. The handling of the tissues at operation usually results either in a decrease or, more often, in an increase of the interstitial spaces which become filled with fluid, blood, or even germ cells (paper I). It is, therefore, difficult if not impossible to use the volume of interstitial space as an iudication of fibrosis. On the other hand, there are cases-and several were found iu our materialill which the fibrous tissue in the interstitium and particularly around the vessels is greatly increased, although there is no appreciable increase in the tunica propria. This type of fibrosis appears to be different from that implied by a large volume of basement membrane and tunica propria. Our data can, therefore, not be used to. quantitate "general fibrosis" within the testis.
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In the present paper we have attempted a quantitative analysis of testicular biopsy specimens, and from our results we draw the conclusion that Chalkley's method is a useful tool in such attempts. Within limits which we have tried to define, the method gives detailed information through relatively simple procedures. We have applied it, however, in less than optimal form because no attempt was made to obtain absolute values concerning tissue volumes. In future investigations it should be possible to repair this omission by taking a few simple, external measurements of the gonad at the time of biopsy and estimating from these the absolute volume of the testes. 7 In this way the Chalkley method could be made to yield absolute data concerning the mass of germinal epithelium, Leydig cells, etc. It is very clear to us that all conclusions drawn from our data are subject to the limitations of our normal standards of comparison. The original normal series was small but carefully selected for "quality" of the testicular tissues. Experience with the present series, which contains six times as many specimens, makes us feel that the standard of comparison is probably too "good"; that a random series of healthy men would show lower means for germ cell volumes, and probably for Leydig cells. Quite certainly the ranges of "normalcy" are greater by 10-20 per cent than those given in paper I, and this must be taken !nto consideration in any future work on this subject.
SUMMARY Chalkley's method for determining the relative volume of tissue components in histologic sections was applied to 180 human testicular biopsies from 125 men with various conditions, among them infertility with azospermia, oligospermia, and normospermia. Results for various categories of cases have been presented and briefly discussed. The data obtained lead to the following classification of the cases with deficient spermatogenesis studied in this series: ( 1) "Late spermatogenic or spermatid arrest." This occurred in more than half of the specimens. It was identified by a disproportional decrease in the volume of spermatids and spermatozoa. ( 2) "Early spermatogenic or spermatocytic arrest" ( 10 per cent of the cases). This was recognized by a disproportional decrease in volume of spermatocytes, spermatids, and spermatozoa. ( 3) "Simple or proportional hypoplasia" (less than 10 per cent of the cases). This was characterized by a proportional decrease of all generations of germ cells. Volumes of Leydig cells were in the average decreased but in individual
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cases the correlation between Leydig cell volume and spermatogenic deficiency was very poor. Volumes of basement membrane and tunica propria were in the average increased in most categories of cases. Such an increase was usually, but not always, associated with subnormal spermatogenesis. It was pointed out that an increase in the bulk of the immediate envelopes of the seminiferous tubules is an indication of only one type of fibrosis occurring in the testis. Sperm counts from 1-105 millionjcc. correlated poorly with the degrees of deficiency of the germinal epithelium. All cases with counts from "very few" to 0.9 million/cc. had greatly diminished germ cell volumes. • The usefuloess and limitations of Chalkley's method in evaluating testicular biopsies have been discussed. REFERENCES 1. CHALKLEY, H. W. ]. Nat. Cancer Inst. 4:47, 1943. 2. CluRNY, C. W. In Diagnosis in Sterility. Proceedings of the Conference on Problems of Human Sterility, 1946. 3. ENGLE, E. T. Ann. New York Acad. Sc. 55:703, 1952. 4. NELSON, W. 0. lo Studies on Testis, and Ovary, Eggs and Sperm. Proceedings of the Conference Committee on Human Reproduction, 1952. 5. NELSON, W. 0., and STEINBERGER, E. Anat. Rec. 112:367, 1952. 6. NELseN, W. 0., and BuNGE, R. G. ]. U