Intratubular germ cell neoplasia of unclassified type occupying the whole testis accompanied by a small mature teratoma and metastatic choriocarcinoma and Sertoli cell-only tubules in the other testis

A 19-year-old man with mild mental retardation was diagnosed as having metastatic choriocarcinoma and a testicular tumor. Histopathological examination of the resected testis revealed the presence of a small lesion of mature teratoma but no trace of choriocarcinoma. The remaining seminiferous tubules were atrophic and lined by large atypical germ cells, which were diagnosed as intratubular germ cell neoplasia of the unclassified type (IGCNU). A small area with prominent tubules was also observed. Within this lesion, the tubules were dilated and contained several layers of cells with central necrosis. Immunohistological comparison of staining for several biological markers (Ki-67, c-kit and placental alkaline phosphatase) between cells in the atrophic tubules and those in the dilated tubules indicated a progression of the latter cells to cells with a more proliferative ability. In the opposite testis, examined at autopsy after death due to metastatic choriocarcinoma, all seminiferous tubules were lined by Sertoli cells only. It was therefore assumed that the germ cell tumor of the combined histological type had primarily arisen in the background of IGCNU, and that choriocarcinoma had spontaneously regressed. The early onset of these testicular neoplastic lesions strongly indicates their occurrence under the genetic background of gonadal dysplasia, the Sertoli cell-only syndrome. The possible relation of gonadal disease to mental retardation in this patient is also discussed.     

Association between testicular dysgenesis syndrome (TDS) and testicular neoplasia: evidence from 20 adult patients with signs of maldevelopment of the testis

Based on a well established association between testicular cancer and undescended testis and more recent publications on epidemiological links between these disorders and male infertility, we proposed the existence of a testicular dysgenesis syndrome (TDS). In most cases TDS presents with impaired spermatogenesis, only in rare cases the full range of its signs, including genital malformations and testicular cancer can be seen in one patient. In order to further corroborate our hypothesis about the presence of testicular dysgenesis in patients with testicular abnormalities, we decided to re-analyse recent testicular biopsies derived from patients with infertility, hypospadias and undescended testis. We searched for histological signs of testicular dysgenesis: microliths, Sertoli-cell-only tubules, immature seminiferous tubules with undifferentiated Sertoli cells, and tubules containing carcinoma in situ (CIS) cells. We identified 20 patients who fulfilled the histological criteria for testicular dysgenesis, 9 of whom were diagnosed with uni- or bilateral testicular germ cell neoplasia, and the remaining ones with subfertility. The presence of CIS was detected in 5 patients (3 of them with overt contralateral germ cell tumours). In all but one of the CIS cases, at least one additional sign of testicular dysgenesis was detected. Clinical records of all patients were subsequently analysed. The majority of cases had oligozoospermia or azoospermia. Their reproductive hormone profiles correlated with the results of semen sampling and testicular histology. In conclusion, our study of 20 patients with various reproductive abnormalities provided evidence that TDS is a real clinical entity. We speculate that most of these abnormalities are caused by adverse environmental effects rather than specific gene mutations.     

COMMENTS

Based on a well established association between testicular cancer and undescended testis and more recent publications on epidemiological links between these disorders and male infertility, we proposed the existence of a testicular dysgenesis syndrome (TDS). In most cases TDS presents with impaired spermatogenesis, only in rare cases the full range of its signs, including genital malformations and testicular cancer can be seen in one patient. In order to further corroborate our hypothesis about the presence of testicular dysgenesis in patients with testicular abnormalities, we decided to re-analyse recent testicular biopsies derived from patients with infertility, hypospadias and undescended testis. We searched for histological signs of testicular dysgenesis: microliths, Sertoli-cell-only tubules, immature seminiferous tubules with undifferentiated Sertoli cells, and tubules containing carcinoma in situ (CIS) cells. We identified 20 patients who fulfilled the histological criteria for testicular dysgenesis, 9 of whom were diagnosed with uni- or bilateral testicular germ cell neoplasia, and the remaining ones with subfertility. The presence of CIS was detected in 5 patients (3 of them with overt contralateral germ cell tumours). In all but one of the CIS cases, at least one additional sign of testicular dysgenesis was detected. Clinical records of all patients were subsequently analysed. The majority of cases had oligozoospermia or azoospermia. Their reproductive hormone profiles correlated with the results of semen sampling and testicular histology. In conclusion, our study of 20 patients with various reproductive abnormalities provided evidence that TDS is a real clinical entity. We speculate that most of these abnormalities are caused by adverse environmental effects rather than specific gene mutations.     

Androgen insensitivity syndrome: an immunohistochemical, ultrastructural, and morphometric study

OBJECTIVE: To evaluate the morphometric, immunohistochemical, and ultrastructural lesions of the testes in prepubertal and adult patients with androgen insensitivity syndrome. METHODS: We examined the testicular biopsy using immunohistochemistry for vimentin, smooth muscle actin, and collagen IV antigens. Quantification of seminiferous tubules and testicular interstitium was performed in prepubertal and adult patients with androgen insensitivity syndrome and results were compared with normal testes from both infants and adults. RESULTS: The adult testes presented nodular and diffuse lesions that consisted of Sertoli-cell-only seminiferous tubules. Two types of Sertoli cells could be distinguished, namely, immature vimentin-positive Sertoli cells and nearly mature Sertoli cells. In the nodules, the lamina propria was thin and contained a scant number of actin-positive peritubular cells. Leydig cells were hyperplastic. The prepubertal patients showed only diffuse lesions characterized by Sertoli cell hyperplasia, decreased germ cell numbers, and a discontinuous immunoreaction to collagen IV. CONCLUSIONS: The testicular lesions in androgen insensitivity syndrome are probably caused by primary alterations that begin during gestation. These lesions become progressively more pronounced at puberty, when the nodular lesion pattern (adenomas) is completely developed.     

The contribution of RNA-binding motif (RBM) antibody to the histopathologic evaluation of testicular biopsies from infertile men

Testicular biopsies of infertile men are often characterized by mixed histologic patterns, with different types of spermatogenic impairments being found in adjacent seminiferous tubules. RNA-binding motif (RBM) is a nuclear protein expressed exclusively in the male germ cell line. We reasoned that RBM might be a useful marker to identify germ cells in testicular sections, particularly in biopsies with mixed histologic phenotype and small focal concentrations of spermatogenesis. Testicular biopsies from azoospermic men were immunohistochemically evaluated for RBM expression. RBM expression was detectable in spermatogonia, spermatocytes, and round spermatids in biopsies of men with obstructive azoospermia and normal spermatogenesis. No specific cell staining was shown in cases of Sertoli-cell-only (SCO) syndrome. In biopsies of patients with spermatogenic disorders, all the germ cells were stained up to and including the stage level of the arrest in spermatogenesis. This approach enabled identification of small focal concentrations of spermatogenesis in a biopsy previously classified as being SCO by hematoxylin and eosin staining. Thus, RBM can be a useful immunohistochemical marker for the specific identification of germ cells and provide greater accuracy in the histopathologic evaluation of testicular biopsies.     

Intratubular germ cell neoplasia

Intratubular germ cell neoplasia is now well established as a precursor to invasive germ cell tumors of the testis. The lesion is usually associated with subsequent development of typical invasive tumors if orchiectomy is not performed. Pathologic findings in intratubular neoplasia consist of germ cell abnormalities within the seminiferous tubules. The atypical cells have large round nuclei, prominent nucleoli, and abundant clear cytoplasm. They have ultrastructural and immunohistochemical characteristics of spermatogenic precursor cells present in the fetal and postnatal testis. Intratubular collections of abnormal germ cells have been found adjacent to invasive germ cell tumors in a high proportion of cases. Intratubular neoplasia occurring in the absence of testicular enlargement has been found in the following situations: cryptorchidism, in testes of some infertile men, dysgenetic testes, and in the contralateral testis following orchiectomy for germ cell cancer. Recognition of intratubular germ cell neoplasia is of special importance because detection of the most common forms of testicular cancer can be accomplished at a preinvasive stage and, therefore, prior to the opportunity for metastasis.     

Mixed testicular germ cell tumor in an adult with cryptorchidism and Down's syndrome

We here present a case of mixed testicular germ cell tumor in an adult with cryptorchidism and Down's syndrome. A 20-year-old Japanese man with a mass in the left inguinal region underwent orchidectomy as a left testicular tumor was suspected. Histology showed a mixed germ-cell tumor with embryonal carcinoma and yolk sac tumor with syncytiotrophoblastic giant cells occurring in a cryptorchid testis. Chromosomal analysis of peripheral lymphocytes disclosed a karyotype of 47,XY,+21[20]. Our case provides further evidence that these three conditions-Down's syndrome, cryptorchidism and testicular germ cell tumor-may be closely associated. To our knowledge this is the first case of mixed germ cell tumor arising in a patient with Down's syndrome and cryptorchidism.     

Absence of RBM expression as a marker of intratubular (in situ) germ cell neoplasia of the testis

RBM (RNA-binding motif) protein is a marker of male germ cells. This protein is encoded by the Azoospermia factor region-b (AZF-b) of the human Y chromosome and is expressed exclusively in the male germ cell line, that is, spermatogonia, spermatocytes, and round spermatids. The authors analyzed the expression of the RBM gene in germ cell tumors and in the seminiferous tubules in the vicinity of these tumors to identify the presence of IGCN. Sections from testicular germ cell tumors of 21 patients were stained with anti-RBM antibody by using an immunohistochemical method. Distal tubules showing spermatogenesis were immunopositive for RBM protein. All of the germ cell tumors studied were completely immunonegative for RBM. Defined areas of IGCN also showed an absence of RBM expression. Tubules with spermatocyte-like cells, which were expected to express RBM, did not express this protein. This result enabled the identification of tubules as being IGCN. RBM is a novel marker consistently expressed in normal male germ cells but not in malignant germ cell tumors or IGCN. Thus, the absence of RBM expression in germ cells provides a new diagnostic tool of preinvasive malignancy of the testis.     

Histological evidence of testicular dysgenesis in contralateral biopsies from 218 patients with testicular germ cell cancer

This study was prompted by a hypothesis that testicular germ cell cancer may be aetiologically linked to other male reproductive abnormalities as a part of the so-called 'testicular dysgenesis syndrome' (TDS). To corroborate the hypothesis of a common association of germ cell cancer with testicular dysgenesis, microscopic dysgenetic features were quantified in contralateral testicular biopsies in patients with a testicular germ cell tumour. Two hundred and eighty consecutive contralateral testicular biopsies from Danish patients with testicular cancer diagnosed in 1998-2001 were evaluated retrospectively. Two hundred and eighteen specimens were subsequently included in this study, after 63 patients who did not meet inclusion criteria had to be excluded. The presence of carcinoma in situ (which is believed to originate from transformed gonocytes) was detected in 8.7% of biopsies. The incidence of other dysgenetic features was immature tubules with undifferentiated Sertoli cells, 4.6%; microcalcifications (microliths), 6.0%; and the presence of a Sertoli-cell-only pattern in at least a few tubules, 13.8%. The cumulative incidence of one or more signs of testicular dysgenesis was 25.2%. In a few patients, areas with immature and morphologically distorted tubules were also noted. Spermatogenesis was qualitatively normal in 51.4%, whereas 11.5% had very poor or absent spermatogenesis. It is concluded that microscopic testicular dysgenesis is a frequent feature in contralateral biopsies from patients presenting with testicular germ cell neoplasms of the adolescent and young type. The findings therefore support the hypothesis that this cancer is part of a testicular dysgenesis syndrome. The presence of contralateral carcinoma in situ was higher in the present study than previously reported.     

Testicular development in cynomolgus monkeys

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.     

Germ cell loss in Klinefelter syndrome: When and why?

Klinefelter syndrome (KS) is a quite common disorder with an incidence of 1–2 in 1,000 new‐born males. Most patients are diagnosed in the light of a clinical checkup when consulting a fertility clinic with an unfulfilled child wish. Infertility in KS patients is caused by a massive germ cell loss, leading to azoospermia in more than 90% of the adult patients. Most seminiferous tubules in the adult KS testis are degenerated or hyalinized and testicular fibrosis can be observed, starting from puberty. However, focal spermatogenesis can be found in the testis of some patients. This offers the opportunity to extract spermatozoa from the testis by testicular sperm extraction (TESE). Nevertheless, TESE is only successful in about half of the KS adults seeking to father children. The reason for the germ cell loss remains unclear. To date, it is still debated whether the testicular tissue changes and the germ cell loss seen in KS is directly caused by an altered X‐linked gene expression, the altered somatic environment, or a deficiency in the germ cells. In this review, we provide an overview of the current knowledge about the germ cell loss in KS patients.     

Sertoli cell types in the Sertoli-cell-only syndrome: relationships between Sertoli cell morphology and aetiology

Histological study of testicular biopsies from infertile men showing Sertoli-cell-only tubules due to hypogonadotropic hypogonadism, cryptorchidism, oestrogen treatment, chemotherapy or Del Castillo's syndrome, revealed four types of Sertoli cells: (1) normal adult mature cells showing an indented nucleus, grossly triangular in shape with a prominent tripartite nucleolus; (2) immature cells with round regularly outlined nuclei and immature cytoplasm; (3) dysgenetic cells showing immature nuclei and a nearly mature cytoplasm with less developed cytoplasmic organelles; and (4) involuting cells with very irregularly outlined nuclei and a mature cytoplasm containing abundant lipid droplets and residual bodies and atypical inter-Sertoli junctional specializations. Testes from men with hypogonadotropic hypogonadism showed only immature Sertoli cells; cryptorchid testes showed dysgenetic cells and occasional normal cells; and after treatment with oestrogens or chemotherapy the testes showed involuting cells and normal cells. The testes of men with Del Castillo's syndrome could be classified into three groups, according to the Sertoli cell type present: mature, dysgenetic and involuting cells. This finding suggests that Del Castillo's syndrome may be due to at least three different aetiologies.     

Microinvasive germ cell tumor of the testis

Microinvasive germ cell tumor (MGCT) consists of a limited number of malignant germ cells in the intertubular tissue of the testis. The cells have large nuclei, prominent nucleoli, abundant clear cytoplasm, and distinct cellular borders in hematoxylin and eosin staining. MGCT can be the first stage of malignancy in the development of testicular germ cell tumor (TGCT). Biopsies from men with maldescended testes have been reported to contain intratubular germ cell neoplasia, unclassified (IGCN) and MGCT in 1.8% of the examined cases (95% CI 0.5–4.6%). MGCT has also been found in testes of subfertile men and in the contralateral testis of patients with TGCT. MGCT is a frequent finding (19%) in the testicular tissue adjacent to an overt TGCT. Men with a high risk of TGCT may gain from screening for precursor lesions of TGCT with ultrasonography of the testes followed by a testicular biopsy if suspicious abnormalities are found: Treatment is high-voltage radiotherapy for intratubular germ cell neoplasia (IGCN), and orchidectomy for MGCT and germ cell tumor in situ, either intratubular seminoma or intratubular embryonal carcinoma. After local treatment, patients with precursor lesions can be followed with a surveillance program. The mRNA levels of invasion-related genes were evaluated based on a DNA microarray data set, and we found two gene abnormalities most relevant for the invasion of malignant germ cells: matrix metalloproteinase 9 (MMP9) and plasminogen activator, urokinase (PLAU) genes were up-regulated in a study comparing tissue samples of TGCT and IGCN.     

Macro-orchidism: light and electron microscopic study of four cases.

A hormonal and quantitative light microscopy study of one man with macro-orchidism associated with mental retardation and fragile X chromosome (case no. 1) and three men with idiopathic macro-orchidism (cases no. 2 to 4) is reported. Hormonal study revealed slightly increased follicle-stimulating hormone serum levels in cases no. 1 to 3. The testes from cases no. 1 (orchidoepididymoectomy specimen) and 2 (testicular biopsy) presented interstitial edema and three different tubular patterns that were arranged in a mosaic-like manner. Type I tubules had an increased diameter (less than 220 microns), dilated lumen, and thin seminiferous epithelium usually consisting of Sertoli cells, spermatogonia, primary spermatocytes, and sometimes a few spermatids. Type II tubules had a normal diameter (180 to 220 microns) and germ cell development varied between complete spermatogenesis and Sertoli-cell-only tubules. Type III tubules had decreased diameter (less than 180 microns), atrophic seminiferous epithelium, and thickened tunica propria. The appearance of the nuclei of the Sertoli cells in the three types of tubules could be either mature or immature. Some of the mature Sertoli cells presented a granular cytoplasm. A few of these granular cells grouped together, forming nests that protruded into the tubular lumen. The testicular biopsies from cases no. 3 and 4 only presented type II tubules that contained both mature and immature Sertoli cells. Quantitative study revealed that the large testicular size was principally due to an increased tubular length in all four cases. Although the seminiferous tubule lesions and interstitial edema suggest an obstructive process, the testicular excretory ducts (studied in case no. 1) appeared normal or only slightly dilated. It is possible that the seminiferous tubule lesions (dilated lumen and germ cell depletion) might be secondary to the Sertoli cell lesions (granular cytoplasm and nuclear immature-like pattern.     

Atypical germ cells of the testis

Summary It is uncertain whether the so called intratubular atypical germ cells (carcinoma in situ cells) demonstrable in the testicular tissue around different germ cell tumors and in testicular biopsies of patients with impaired fertility are identical with regard to their morphology and further development. Thus atypical germ cells of 18 patients with testicular germ cell tumors and of 3 patients with atypical germ cells in testicular biopsies without tumor were studied by electron microscopy and/or by immunohistochemistry. The atypical germ cells show characteristic alterations distinguishing them from normal germ cells, especially spermatogonia. However, there are no differences between atypical germ cells in the above mentioned groups. Immunohistochemical reactions are negative with anti-alpha-fetoprotein and anti-beta-human-chorionic-gonadotropin, but 6 of the 15 cases are positive with antiferritin. However, this positive reaction occurs in cases in different diagnostic groups. Atypical germ cells of the different groups cannot be distinguished by electron microscopy or immunohistochemical methods, but further investigations vestigations, including cell cultures, may provide more information.     

Interstitial compartment pathology and spermatogenic disruption in testes from impotent diabetic men

Studies utilizing animal models of diabetes suggest that diabetic complications of impotence involve structural lesions in the testis as part of an overall defect in the pituitary-testicular axis. In the present study testicular biopsies from ten oligospermic and/or impotent men with diabetes were evaluated by light and electron microscopy. One biopsy was judged normal. The remaining tissue showed variable testicular pathology ranging from minimally to grossly affected. Seminiferous tubules had decreased tubule diameters, hyalinized tubule walls, and occluded lumina owing either to epithelial encroachment or cellular debris and exfoliated round germ cells. Sertoli cells were vacuolated and showed a high degree of apical cell membrane redundancy and degeneration. Although Sertoli-Sertoli cell junctional complexes appeared normal, Sertoli junctional specializations associated with spermatids were structurally abnormal or absent. All tubules were variably depleted of adluminal compartment germ cell types. The interstitial compartment was filled with a collagen-rich extracellular matrix concentrated around small blood vessels and seminiferous tubule walls. Capillaries and lymphatic endothelia appeared structurally abnormal and compromised by the interstitial "matrix expansion." Some Leydig cells contained a variable number of small to large lipid droplets, vacuoles, and secondary lysosomes. Results indicate the presence of tissue pathology in testes of impotent diabetic men. Discrete ultrastructural lesions in apical Sertoli cell cytoplasm are associated with spermatogenic disruption and morphological changes in the interstitial compartment suggest microvascular complications.     

A case of primary testicular germ cell tumor with rhabdomyosarcoma metastases as an example of applying the FISH method to diagnostic pathology

We present the interesting case of a 38-year-old man with a primary malignant tumor of the right testis that metachronously metastasized to the urinary bladder and the stomach. Histologically, the testicular tumor was a mixed germ cell tumor composed of teratoma and embryonal carcinoma, but it also contained a sarcoma component of somatic type malignancy. Metastases showed rhabdomyoblastic differentiation histologically identical to the sarcoma component of the testicular tumor that was diagnosed as rhabdomyosarcoma. By applying fluorescence in situ hybridization (FISH) to the cytogenetic examination of cells taken from the periventricular lymph node metastases, we demonstrated a structural chromosomal aberration characteristic of testicular neoplasms, i.e. the presence of isochromosome 12p (i(12p)). Additionally, the diagnosis was supported by immunohistochemistry.     

Morphometric study of the prepubertal rabbit testis: Germ cell numbers and seminiferous tubule dimensions

Testes from rabbits aged 1–9 weeks were examined by light microscopy. Changes in seminiferous tubule dimensions, testicular volume, and volume fraction of tubules were assessed. Germ cells and Sertoli cells were counted in round tubular cross sections and total germ cell number in each testis was estimated. Mitotic, meiotic, and degenerative activities of germ cells as well as their basal or central positions within tubules were quantified. A marked, steady increase in testis volume and in tubular length and volume occurred over the prepubertal period; but diameter underwent no significant increase and in fact decreased until week 4. Overall, tubules lengthened 40-fold and testis volume increased 25-fold; the percentage volume of the testis occupied by tubules rose from one-third neonatally to three-fifths at the onset of spermatogenesis. The ratio of germ cells to total tubular (germ and Sertoli) cells was lowest at 3 weeks. However, the total number of germ cells increased little until 3 weeks, after which it rose at a sharp rate commensurate with testis volume. Percentage of germ cells in mitosis peaked sharply at 3 weeks, dropped in subsequent weeks, and then rose at 7 weeks at the initiation of spermatogenesis. Importantly, the surge in mitosis at 3 weeks was followed by a redistribution of germ cells to a predominantly basal location from 3 to 7 weeks. Meiotic activity was sparse at 7 weeks and became abundant by 9 weeks. Germ cell degeneration remained relatively constant during weeks 1 through 6, with an increase at 7 weeks.     

Multinucleated spermatogonia in cryptorchid boys: A possible association with an increased risk of testicular malignancy later in life?

At birth, undescended testes contain germ cells, but after 1 year of life, a reduced number of germ cells is generally found. Microlithiasis and carcinoma-in-situ-testis occur in cryptorchid boys. Multinucleated germ cells, including at least 3 nuclei in the cell, exist in impaired spermatogenesis and in the senescent testis. AIM OF THE STUDY: We investigated whether multinucleated spermatogonia were present in undescended testes of cryptorchid boys, and if such a pattern is associated with special clinical features. RESULTS: Multinucleated spermatogonia occurred in 13/168 (8%) of 163 consecutive cryptorchid boys, who underwent surgery for cryptorchidism with simultaneous testicular biopsy showing seminiferous tubules. The patients with multinucleated spermatogonia more often exhibited a normal germ cell number (Fisher's exact test, p<0.0005), and were younger at surgery (Mann Whitney, p<0.005) than the rest of the patients. Before surgery, 3 patients underwent treatment with Erythropoietin because of renal failure. An intra-abdominal testis underwent clipping and division of the spermatic vessels, and a biopsy at final surgery 7 months later, exhibited multinucleated spermatogonia. In 1 case the undescended testicular position, a fixed retraction, was acquired after surgery for an inguinal hernia. Multinucleated spermatogonia were found in cases of carcinoma-in situ-testis in 2 cryptorchid boys. No case of multinucleated germ cells appeared in our normal material. CONCLUSION: Multinucleated spermatogonia are a further abnormality present in cryptorchidism. The cryptorchid boys with multinucleated spermatogonia in general exhibited rather many germ cells. This feature may be associated with an increased risk of testicular malignancy later in life, and we propose a careful follow up regime in these cases including ultrasound examination and a testicular biopsy in cases of symptoms or clinical findings.