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From the * Department of Morphology, School of
Medicine, Autonomous University of Madrid, Madrid; the
Department of Pathology, La Paz Hospital,
Madrid; and the Department of Cell Biology and
Genetics, University of Alcalá, Alcalá de Henares (Madrid),
Spain.
| Correspondence to: Dr M. Nistal, Department of Morphology, School of Medicine, Autonomous University of Madrid, E-28029 Madrid, Spain. |
| Received for publication September 25, 2002; accepted for publication March 17, 2003. |
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Abstract |
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Key words: Male infertility, testicular biopsy, germ cell quantitation
Comparison of the germ cell values calculated in testicular biopsies with control values permits the evaluation of the spermatogenetic capacity of the testes. The number of adult spermatids (Sc+Sd) in the testis can be compared with spermatozoa in the spermiogram by means of a power curve that correlates both sets of data (Silber and Rodriguez-Rigau, 1981; Nistal et al, 1987). These comparisons have revealed that sperm excretory duct obstruction is a frequent cause of azoospermia and severe oligozoospermia (Galmes-Belmonte and Nistal, 1998). If the absence of spermatozoa in the spermiogram is associated with the presence of adult spermatids in testicular biopsy, such azoospermia has an obstructive cause. If testicular biopsy reveals more adult spermatids than spermatozoa in the ejaculate, the oligozoospermia is considered obstructive.
Previous studies of patients with obstructive azoospermia seen in our hospital have shown that the most frequent lesions occur in the adluminal compartment of the seminiferous epithelium, which showed more adult spermatids than young spermatids. This finding is surprising because, in normal testes, the number of adult spermatids is about 23.3% lower than that of young spermatids (Nistal and Paniagua, 1997). The aim of this study was to evaluate the frequency of this finding and provide an explanation for it.
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Materials and Methods |
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In the 22 men selected for the study, the causes of azoospermia were: vasectomy performed between 3 and 17 years before biopsy (7 patients); bilateral agenesis of the vas deferens (3 patients); bilateral epididymal enlargement associated with respiratory diseases (Young syndrome, 3 patients), bilateral cysts in the caput epididymidis (1 patient), bilateral inguinal herniorrhaphy (1 patient), left varicocele (1 patient), and unknown causes (normal physical exploration, 6 patients). Before and after surgery, the 22 patients presented normal basal serum levels of follicular stimulating hormone (FSH), luteinizing hormone (LH), testosterone, prolactin, and inhibin B. Testicular and semen volumes and levels of both fructose and citric acid were also normal.
Testicular biopsy was bilateral in 19 patients and unilateral in 3 patients: case 3, a vasectomized man with history of left orchidoepidydimoectomy owing to testicular trauma; case 11, a man who lost his left testis because of neonatal torsion; and case 22, a bilaterally biopsied man whose left testis biopsy was poorly fixed and was excluded from the study (Table 1). Therefore, a total of 41 biopsies were available.
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The study also included unilateral testicular biopsies from three patients (aged 18, 21, and 23 years) who presented because of testicular torsions of less than 8 hours of evolution, and because the quantitative study of biopsies from the removed twisted testes revealed that the number of adult spermatids was higher than young spermatids (Table 1).
All testicular biopsies were fixed in Bouin solution for 24 hours and embedded in paraffin. Eight 6-µm thick, nonconsecutive sections were obtained from each testis, mounted on the same slide, and stained with hematoxylin-eosin. In each of these sections, the number of spermatogonia, primary spermatocytes, young spermatids (Sa + Sb), and adult spermatids (Sc + Sd) were counted in 20 cross-sectioned seminiferous tubules per section, and the average values were calculated. Spermatid classification was done according to Clermont's criteria (Clermont, 1963). Testicular biopsies obtained 6 to 8 hours after death from 10 males (20 to 49 years of age), who died because of myocardial infarct or in traffic accidents, had not suffered from testicular or related diseases, and had proven fertility were used as controls (Nistal et al, 1987). These control values were similar to those reported by other authors (Johnsen, 1970; Skakkebaek and Heller, 1973; Skakkebaek et al, 1973; Makler and Abramovici, 1978; Silber and Rodriguez-Rigau, 1981; Nistal and Paniagua, 1997). According to these controls, the lowest limits of normal testicular parameters were the following: 17 spermatogonia, 25 primary spermatocytes, 30 young spermatids, and 23 adult spermatids. This means that the number of adult spermatids was 23.3% lower than the number of young spermatids: (30-23)100/30. In addition, the average tubular diameter was evaluated in the same cross-sectioned tubules (the lowest normal value was 185 µm).
In order to determine whether the etiology of the obstruction was related to the evaluated parameters, testicular biopsies were classified into 3 groups: vasectomized patients (7 men, 13 testes), other causes of obstructive azoospermia (15 men, 27 testes), and testicular torsion (3 men, 3 testes) (Table 1).
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Results |
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Of the 44 biopsies, germ cell development was observed in 42 testes. In one patient (case 13), the left testis showed only completely hyalinized seminiferous tubules, and another patient (case 14) showed Sertoli cell-only tubules in his left testis. The results of germ cell quantitations are shown in Tables 1 and 2, and the Figure.
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The 7 vasectomized men showed more adult spermatids than young spermatids in one (6 men) or both (1 man) testes. This increase varied widely among testes, from 0.5 cells (3.2%) to 20.4 cells (130%) per cross-sectioned tubule, with an average value of 8.3 cells (51.4%). Since in normal seminiferous tubules the number of adult spermatids is 23.3% that of young spermatids, the actual increase that should be assumed for azoospermic men would vary from 21.5% (3.2 + 23.3) to 153.3% (130 + 23.3), with an average value of 68.7% (Tables 1 and 2, and the Figure).
Of the 27 biopsies from the 15 men with obstructive azoospermia due to causes other than vasectomy, increased numbers of adult spermatids in relation to young spermatids were found in 22 testes. Obstructive azoospermia was bilateral in 7 of the 12 patients whose bilateral biopsies were available for the study. The differences between the number of adult spermatids and the number of young spermatids varied from 0.4 cells (1.2%) to 11.6 cells (264.2%) per cross-sectioned tubule, with an average value of 5.1 cells (43.6%) per cross-sectioned tubule. The corrected percentage (by adding 23.3%) varied from 24.5% to 287.5% (average 66.9%) (Tables 1 and 2, and the Figure).
The biopsies of 3 twisted testes showed characteristic lesions of degree II hemorrhagic infarct, according to Mikuz's classification (Mikuz, 1985). The increases in the numbers of spermatids during maturation were 2.8 cells (20.5 %), 24.4 cells (57.5%), and 3.8 cells (9.6%), respectively, with an average value of 29.2% (52.5% after correction) (Tables 1 and 2, and the Figure).
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Discussion |
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This finding poses 3 questions: 1) What is the cause of such increase? 2) What are the differences among patients or between both testes within the same patient? and 3) What is the reliability of the power curves that correlate germ cell numbers in testicular biopsy and sperm numbers in spermiogram, in order to evaluate obstructive azoospermias?
A higher ratio of adult-to-young spermatids has been observed in testosterone-treated rats; this has been interpreted as failure of the spermiation process, caused by the transient drop in testosterone levels (Saito et al, 2000). This was not the hormonal status of the patients studied here, who presented normal levels of gonadotropins and testosterone. In any case, a hormonal change would impair both testes in a similar way. The most probable explanation is a stagnation of testicular fluid, caused by sperm excretory-duct obstruction. Obstruction of the proximal segments of the excretory duct (epididymis and proximal portion of the ductus deferens) in both laboratory animals (Smith, 1962; Lopez et al, 1988) and humans (Nistal and Paniagua, 1999) has been shown to produce the histological testicular lesion known as "obstructive pattern." These lesions start when the capacity of the ductuli efferentes to reabsorb testicular fluid is exceeded; and the closer the obstruction is to the testis, the more severe the lesion. Our 22 azoospermic patients presented this characteristic obstructive histological pattern and, although the anatomical level of obstruction could not be determined in the group of vasectomized patients, the similarity of the histological lesions among the different groups of azoospermic patients studied, together with the absence of data supporting a distal obstruction, suggests that the lesion mechanism is the same in the 22 patients. This conclusion is also supported by the observation of the same findings in the 3 twisted testes. Most adult testicular torsions are intravaginal as torsion involves all structures of the spermatic cord, which becomes surrounded by a high insertion of the tunica vaginalis testis, causing strangulation of the veins and sperm excretory ducts. The accumulation of testicular fluid and spermatozoa in the intratesticular ducts (seminiferous tubule lumen and rete testis channels) and the extratesticular ducts near the testis, caused by obstruction makes spermiation difficult. As a result, adult spermatids accumulate in the seminiferous epithelium.
The second question is: Why is the lesion not always bilateral and not present in all patients with obstructive azoospermia? Only 1 of the 7 vasectomized patients who underwent bilateral testicular biopsy showed an increased number of adult spermatids bilaterally. There are 2 explanations for the unilateral increase in adult spermatids: 1) differences in the surgical procedure, and 2) the obstruction being followed by reactions that reduce the increased intratubular pressure by variable degrees. The first explanation is not likely in the vasectomized men studied here because the technique used was always the same (ligation of both ends of the ductus deferens), and, in any case, a surgical procedure would only justify differences among patients, but not between testes from the same patient. Since germ cell counts are similar to those observed in vasectomies using the open proximal end technique (Moss, 1992; Whyte et al, 1998), the explanation seems to be related to the occurrence of a spermatic granuloma in the proximal end of the sectioned ductus deferens. The incidence of spermatic granuloma in vasectomized patients varies from 20% to 35% (Belker et al, 1983) to 60% (McDonald, 1966). When the incidence is compared in different vasectomy techniques, the figures vary from 20.8% to 58.4% (McCallum et al, 2000). Therefore, whatever the technique employed, at least 20.8% spermatic granulomas are to be expected. Studies in humans (Lopez et al, 1988), as well as laboratory animals (Silber, 1977; Sheynkin et al, 2000), lead us to conclude that spermatic granuloma causes a decrease in excretory duct pressure. This granuloma would produce, in addition to spermatozoon destruction, reabsorption of the testicular and epididymal fluids. Spermatic granuloma is a frequent finding in vasectomy (Belker et al, 1983; Lopez et al, 1988). The cause of these granulomas is unknown, but it has been proposed that they occur when the ligature is too tight or when cauterization is excessive (Belker et al, 1983).
In the group of nonvasectomized azoospermic patients, bilateral testicular biopsy was performed in 12 patients; the increased number of adult spermatids was bilateral in 7 of them. This higher bilaterality rate might be related either to the occurrence of congenital lesions of the ductus deferens or of the ductus epididymidis observed in many of these patients (congenital absence of ductus deferens, Young syndrome, epididymal cysts, etc.). Although it is well known that the effects of congenital obstruction on the testes are milder than those of acquired obstructions (Hirsch and Choi, 1990), the absence of spermatic granulomas in congenital obstructions would probably account for less sperm destruction, as well as less reabsorption of testicular fluid.
The third question was whether the increased number of adult spermatids casts doubt on the predictive value of germ cell counts with regard to the number of spermatozoa expected in the spermiogram. It should be taken into account that the estimate of spermatozoa on the basis of the number of adult spermatids in testicular biopsy is biased because the increased number of adult spermatids in these azoospermic men does not measure the actual number of spermatozoa produced by these testes. At first sight, the number of young spermatids seems to be more pertinent to such evaluation. However, the predictive value of this number is also doubtful. The reason is that, although most obstructions produce lesions in the adluminal compartment, including the loss of primary spermatocytes, these lesions are reversible as soon as the patency of sperm excretory ducts is restored. This means that the first consequence of obstruction is an increase in the number of young spermatids and, thereafter, of adult spermatids. Only a long-term study of patients with obstructive azoospermia, whose cause has been detected and treated adequately would provide worthwhile information about this question
In summary, differential counting between young spermatids and adult spermatids is a rapid and simple method of diagnosing the presence of an obstruction. This is so even in the absence of clinical data, when the number of adult spermatids is higher than young spermatids. This finding has been observed in 41.5% of patients with obstructive azoospermia and might be bilateral, mainly in patients with congenital obstruction. A finding of fewer adult spermatids than young spermatids is also consistent with an obstructive process if the testes bear lesions in the adluminal compartment of the seminiferous tubules, and these lesions have a mosaic distribution (obstructive pattern).
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Footnotes |
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References |
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