DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

Immunocytochemical staining of fine-needle aspiration biopsies of the liver as a diagnostic tool for hepatocellular carcinoma.

Fine-needle aspiration biopsy (FNAB) under ultrasonographic or computerized tomographic guidance is a useful diagnostic procedure for hepatic neoplasms. However, cytologic criteria alone may not allow for the distinction of hepatocellular carcinomas (HCC) from cholangiocarcinomas (CC) and metastatic adenocarcinomas (MA). In an effort to refine the FNAB diagnosis of hepatic malignancies, a panel of immunocytochemical stains was applied to aspiration specimens from primary and metastatic carcinomas in the liver. Anticytokeratin antibodies with different specificities (Cam 5.2 and AE1) were used in conjunction with antibodies to carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and alpha-1-antitrypsin (AAT). All HCC, CC, and MA were immunoreactive with the antikeratin antibody Cam 5.2. However, only three (15%) HCC were positive with AE1, in contrast to 100% of CC and MA. Antibodies to CEA and AFP were also helpful diagnostic aids, especially for the three HCC that were immunoreactive with AE1. Canalicular staining for CEA was present in 47% of HCC, but in none of the CC or MA. AFP positivity occurred in 45% of HCC, but only one CC and none of the MA. AAT was not a useful marker for HCC due to low sensitivity and specificity. Immunocytochemistry is an effective adjunct to the cytodiagnosis of malignant liver tumors sampled by FNAB.     

Immunohistochemical examination of routinely processed bone marrow biopsies.

Immunohistochemistry was performed on paraffin sections of 169 bone marrow biopsies fixed in a buffered methanol-formalin solution and decalcified with EDTA. The biopsies included specimens with normal hematopoiesis, and specimens that were affected by various hematological disorders as well as some metastatic carcinomas. The results demonstrate that a wide spectrum of antigens was preserved in routinely processed bone marrow biopsies, even after long-term fixation up to 12 days. Markers for granulopoietic cells were lysozyme, elastase, DAKO-M 1, and MT 1. Megakaryopoiesis was stained with glycoprotein IIIa, von Willebrand factor, and Ulex europaeus agglutinin (UEA), and erythropoiesis with LN 1. Normal lymphocytes as well as lymphoma cells of all non-Hodgkin's lymphomas tested were positive for leukocyte common antigen (LCA), and at variable degree, for MB 1, 4 KB 5, LN 1, LN 2, UCHL 1, or MT 1. Reed-Sternberg and Hodgkin's cells in Hodgkin's lymphomas were reactive with Ber-H 2, LN 2 and Dako-M 1. In plasma cell disorders, staining for immunoglobulin light chains gave best results. Metastatic carcinomas showed predominantly staining with EMA, and KL 1. A selected panel of specific cell markers is proposed, which proved to be helpful in routine bone marrow diagnosis in most cases.     

The adolescent varicocele. A histopathologic study of 13 testicular biopsies

Testicular varicocele, the most common cause of male infertility, frequently presents in early adolescence. To determine whether testicular damage occurs early in the natural history of varicocele, testicular biopsy specimens from 13 patients, 13 to 18 years of age (mean age, 15.5 years), were studied. The biopsies were compared with testicular tissue from six normal control subjects 15 to 28 years of age (mean age, 23.2 years). Nine of the patients with varicoceles (69.2%) demonstrated some degree of tubular sclerosis. Ultrastructural study demonstrated that the tubular sclerosis was due to collagen deposition by fibromyocytes in the peritubular sheath. Premature germ cell sloughing was present in greater than 50% of tubules examined in all but one biopsy. Six patients (46%) demonstrated small vessel sclerosis. Quantitation of the germinal epithelium revealed that the mean germ cell/Sertoli cell ratio and the percentage of germ cells present as late stage forms (secondary spermatocytes, spermatids and spermatozoa) were significantly reduced in the varicocele group. The testes of two patients exhibited severe hypospermatogenesis approaching germ cell aplasia. None of these changes were seen in the control group. The authors conclude that pathologic changes in the testes of patients with varicoceles are found at or soon after puberty. The histopathologic features include peritubular sclerosis, small vessel sclerosis, premature germ cell sloughing, and variable degrees of hypospermatogenesis.     

Deeper examination of negative colorectal biopsies

Initial histologic sections of specimens from colorectal biopsies of putative lesions may lack polyps. These sections may contain lymphoid aggregates that seemingly correlate with endoscopic findings; however; additional sections might contain polyps. We reviewed 83 specimens from colorectal biopsies of putative lesions for which initial sections lacked polyps. Our objectives were to determine the incidence of polyps within additional sections and to determine whether the presence of lymphoid aggregates within initial sections excludes the presence of polyps within additional sections. Eight specimens (10%) contained polyps (5 adenomatous, 3 hyperplastic), which remained histologically occult until examination to depths of approximately 120 to 380 microm. Five polyps (62%) were associated with lymphoid aggregates that were present within initial sections. We conclude that additional sections may contain surprisingly large numbers of polyps and that lymphoid aggregates present within initial sections fail to exclude the presence of polyps within additional sections.     

Analyses of meiotic chromosomes in testicular biopsies of infertile patients

Between 1989 and 1992 meiotic chromosome studies and synaptonemal complex analyses were evaluated using light and, in part, electron microscopy in 46 infertile males with highly abnormal spermiograms. This examination focused on whether the breakdown of spermatogenesis could be attributed to pairing anomalies of bivalents. The study of meiotic chromosomes and synaptonemal complexes indicated normal spermatogenesis in five patients (11%); in the remainder, maturation arrest was diagnosed. In 21 individuals (50%) the breakdown was accompanied by pairing anomalies (asynapsis, fragmented synaptonemal complexes, X/Y univalence). Thus it is shown that male infertility can often partly be explained by meiotic disorders.      

Light and electron microscopic analysis of human testicular spermatozoa and spermatids from frozen and thawed testicular biopsies.

The morphological changes caused by freezing and thawing human testicular spermatozoa have been assessed here. Retrieval of testicular biopsies was carried out on six patients with obstructive azoospermia preparatory to intracytoplasmic sperm injection (ICSI). Light microscope analysis was carried out on testicular cells and ultrastructural analysis was carried out on spermatozoa and different spermatid stages before and after the freezing procedure. Upon examination under light microscopy, all germ cells presented increased vacuolization in their cytoplasm and shrinkage or swelling of the nuclei and cytoplasmic membranes. These altered structures were accentuated in the spermatocyte I cell which often presented disrupted membranes. The ultrastructural findings under transmission electron microscopy demonstrated that after freezing and thawing the major types of cryoinjury were the swelling and rupture of inner and outer acrosomal and plasma membranes. The acrosome material often appeared as dispersed material or as condensed spots or was even lost. Such damage was observed mainly at the spermatozoa and late spermatid stages. We conclude that the freezing and thawing of testicular biopsies causes similar morphological damage to testicular spermatozoa and frozen-thawed ejaculated spermatozoa. It is still unclear whether these changes in testicular spermatozoa after freezing and thawing may compromise its use in the ICSI procedure.     

A quantitative approach to the classification of hypospermatogenesis in testicular biopsies for infertility.

Absolute counts of germ cells were performed in 110 testicular biopsy specimens from 59 patients with either idiopathic infertility or varicocele and in a series of five autopsy specimens from age-matched controls. The tubular diameter, thickness of the tubular wall, and density of Leydig cells were measured. The following patterns were identified by germ-cell counts in the biopsy specimens: normal cell population, mild decrease in germ cells with normal ratio of cell types, advanced hypospermatogenesis with abnormal ratio of cell types, and Sertoli cell only. This sequence of progressive hypospermatogenesis was remarkably similar in both series. A separate category of maturation arrest was not recognized. Cell counts also correlated between right- and left-sided samples from the same patient in both series of biopsies. Reduction of tubular diameter, thickening of the tubular wall, and increase in Leydig cell density were often seen in severe stages of germ-cell impairment, although with an irregular distribution.     

The WISC as a diagnostic tool.

Wechsler's hypotheses for the adolescent sociopath were applied to the WISC under controlled conditions. Groups were separated according to race and sex, while IQ, socioeconomic class, and geographic location were held constant. The criterion for selection of Ss was appearance before a juvenile court. The efficiency of each of Wechsler's signs was determined. Post hoc analysis provided specific signs for each combination of race and sex in the 80-89 IQ range. The need for cross-validation of these signs was stressed. Other methodological issues that may contribute to inconclusive findings with traditional assessment procedures were discussed throughout the study. These included: (1) the need for control of the relevant variables; (2) the problem of poorly defined criteria; (3) the need for analysis of the individual as well as the group data; (4) the importance of a holistic approach; (5) the problem with matching; and (6) the problem of not having an expected distribution with which to compare the frequency of "hits" for Wechsler's indices.     

An examination of the variation in timed endometrial biopsies

Endometrial biopsies were obtained from fertile women, aged20–40 years, with regular cycles of 25–35 days.Chronological dating of the material was carried out by determinationof the luteinizing hormone (LH) peak by daily LH assay. Tissuewas taken from three or four sites along the biopsy and processedfor electron microscopy. Half micron thick sections were analysedby light microscopy and a morphometric examination of the glandularepithelium carried out. Nuclear size was assessed using an unbiasedmeasuring technique and the volume density of the nucleus inthe cell was also measured. A series of 12 women was examined,four at each of days LH+2,4 and 5. An attempt was made to controla variety of factors which contribute to the variability inthe histological dating of the endometrial biopsy. In doingso it was possible to analyse the relative contributions ofeach sampling level to the overall variance. In contrast toearlier studies there is a relatively small inter-subject variationwith a correspondingly large within-biopsy variation. Theseresults may indicate that the cellular events in the glandularepithelium, between ovulation and the middle of the luteal phase,are precisely regulated.     

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.     

Histological changes in testicular biopsies from chronic alcoholics with and without liver disease.

Testicular and liver biopsies were obtained from thirty consecutive patients at the age of 30 and 64 years with chronic alcoholism (daily consumption of 72 g of alcohol or more for at least five years). The spermatogenesis was normal in 12 patients (40 per cent), moderately reduced in 15 patients (50 per cent) and severely reduced in 3 patients (10 per cent). No relation between the presence of liver disease as assessed by histological examination of the liver biopsy and the impairment in the spermatogenesis could be demonstrated.     

Maturation phenotype of Sertoli cells in testicular biopsies of azoospermic men

The involvement of Sertoli cells in different spermatogenic impairments has been studied by an immunohistomorphometric technique using cytokeratin-18 (CK-18) as a marker for immature Sertoli cells. CK-18 is known to be expressed in Sertoli cells during prenatal and prepubertal differentiation and is normally lost at puberty. Forty-nine azoospermic men were included in the current study. Quantitative measurements on testicular biopsies revealed the highest CK-18 expression in the mixed atrophy biopsies (22 men), a lower expression in the Sertoli cell-only (SCO) biopsies (12 men), and minimal residual staining in the group considered as representing normal spermatogenesis (six obstructive azoospermia patients). The cytokeratin immunopositive-stained tubules were associated either with arrest in spermatogenesis or with SCO. Examination of sections from nine men with microdeletions in the AZF region of the Y chromosome revealed that these men were either negative for CK-18 expression or showed only weak residual staining. This may suggest that the spermatogenic defect in the AZF-deleted men originates in the germ cell and has no impact on Sertoli cell maturation. The cause that determined the spermatogenic defect in the other cases of male infertility with high CK-18 expression may have damaged both the Sertoli and the germ cells.     

Recommendations for the examination of peripheral nerve biopsies

 Peripheral nerve biopsy is now an established, valuable investigative procedure, but as it can give rise to significant residual symptoms it should only be undertaken after careful consideration of the indications and with informed consent from the patient. Nerve biopsies should only be processed and evaluated in a laboratory with the relevant particular expertise. It is generally recommended that a sural nerve biopsy be performed in combination with a muscle biopsy but not vice versa (muscle biopsies together with a nerve biopsy). Nerve biopsy is not the only means of sampling peripheral nerve tissue to study the peripheral nervous system. Examination of the innervation of the skin may be informative. The same is likely to be true for motor point muscle biopsy. Nerve biopsy is mainly used for morphology although molecular genetic techniques using fresh or archival nerve biopsies are increasingly available. Chemical analysis is undertaken mainly for research purposes. Received: 10 June 1997 / Accepted: 29 October 1997