II.3.9 Evaluation of Testicular Biopsy Samples from the Clinical Perspective

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1 454 Diagnostic Tools.9 Evaluation of Testicular Biopsy Samples from the Clinical Perspective M. Bergmann Summary Testicular biopsy is indicated therapeutically in cases of obstructive azoospermia, and also in cases of hypergonadotrophic testicular azoospermia to recover testicular spermatozoa (testicular sperm extraction = TESE) for assisted reproduction by intracytoplasmic sperm injection (ICSI). A diagnostic biopsy is indicated in cases of refertilization after vasectomy, and also to exclude a testicular tumour. According to European Association of Urology (EAU) guidelines, several testicular samples from three different locations should be taken because of regional differences in spermatogenesis. Histological evaluation is recommended instead of testicular fine needle aspiration (TEFNA). Specimens should be fixed in Bouin s solution to ensure good preservation of tissue structure and to allow the application of modern histological techniques such as in situ hybridization and immunohistochemistry for evaluating gene expression at the mrna and protein levels. Diagnosis of pre-invasive carcinoma in situ (CIS, synonym: testicular intraepithelial neoplasia = TIN) is based on the immunohistochemical demonstration of placentalike alkaline phosphatase (PLAP), which is exclusively expressed in CIS cells. Histological evaluation should be performed using a score count system, determining at least the percentage of seminiferous tubules containing elongated spermatids, which is the most important parameter for TESE/ ICSI, together with a cytological analysis, which gives further causal evidence for the observed spermatogenic impairment. Testicular biopsy is an invasive surgical operation with a high impact on patients with severe spermatogenic impairment, and should therefore be performed only by considering strict criteria of indication, surgical procedure and histological evaluation in qualified centres that are certified, i.e. recommended by the European Academy of Andrology (EAA)..9.1 Indication Testicular biopsy is an invasive diagnostic tool to evaluate spermatogenesis and has to be performed only following strict criteria. It completes history taking, physical examination, scrotal ultrasonography, ejaculate and hormonal analysis. It is indicated in cases of azoospermia, when obstruction of the genital tractissuspectedbecauseofnormaltesticularvolume, normal follicle-stimulating hormone (FSH) levels ( e 7 IU/l), and low levels of epididymal ( [ -glucosidase, L-carnitine) or seminal vesicle (fructose) markers. It should also be performed in the case of refertilization (vaso-vasostomy) after vasectomy or micro-surgical-epididymal sperm aspiration (MESA) to exclude impairment of the seminiferous epithelium. In both cases, cryopreservation of at least part of the biopsy sample offers the opportunity for testicular sperm extraction (TESE) in combination with intracytoplasmic sperm injection (ICSI), if chirurgical refertilization techniques fail. It is also indicated in cases of testicular hypergonadotrophic azoospermia suggested by high levels of FSH ( & 7 IU/l) indicating focal or total Sertoli cell only syndrome (SCO) (Bergmann et al. 1994). In these cases, TESE from remaining focal areas of spermatogenesis within the testis and ICSI form the only rational therapy for assisted reproduction. A diagnosticbiopsyisindicatedincasesofinhomogeneoustesticular ultrasonography to exclude pre-invasive carcinoma in situ (CIS, synonym: testicular intraepithelial neoplasia: TIN) (von Eckardstein et al. 2001). Biopsy of the contralateral testis is indicated when a testicular tumour is clinically evident, because contralateral CIS is reported to have a prevalence of about 5 6%, and of about 2 4% in cases of adult cryptorchidism (Rørth et al. 2000). Testicular biopsy may also be indicated in azoospermic Klinefelter patients, because spermatogenesis, even at a very low rate, might occur allowing TESE and ICSI (Lanfranco et al. 2004). Indications for performing a testicular biopsy are summarized in Table.11. Table.11. Indications for testicular biopsy Inthecaseof Obstructive azoospermia including refertilization after vasectomy (vaso-vasostomy) for TESE/ICSI Hypergonadotrophic azoospermia for TESE/ICSI To exclude testicular tumour Inthecaseof Contralateral testis in the case of unilateral testicular tumour Sonographic testicular microlithiasis Adult cryptorchidism

2 .9 Evaluation of Testicular Biopsy Samples from the Clinical Perspective Preparation Different surgical techniques for obtaining testicular tissue are used. Percutaneous testicular fine needle aspiration (TEFNA) has been recommended for both the assessment of spermatogenesis (Craft et al. 1997) and sperm retrieval in nonobstructive azoospermia (Lewin et al. 1999) assuming a less traumatic nature compared to the main approach of open TESE (Silber et al. 1995). However, it was shown in a controlled animal model system that TEFNA may also produce widespread architectural distortion of seminiferous tubules, especially after repeated punctures, leaving only Sertoli cells (SCO) as well as focal chronic inflammation and necrosis. The latter was also shown to occur after TESE (Shufaro et al. 2002). A technique combining cryopreservation, TESE and histology was first developed by Jezek et al. (1998).Itwaslaterimprovedandpublishedas EAU Guidelines on Male Infertility by Weidner et al. (2002). This technique includes histological evaluation using a scoring system of three biopsies per testis, which are taken from different locations taking account of the testicular vascularization pattern (Fig..47). Two parts of the biopsy sample are cryopreserved to recover spermatozoa for ICSI, and to be stored. The third part is fixed and embedded for histological evaluation, because it offers the opportunity for a causal histological evaluation of spermatogenesis. Formalin fixation of specimens as regularly used in pathology cannot be recommended because of severe shrinkage artefacts, making a detailed histological evaluation impossible (Fig..48a). Fixation in glutaraldehyde, and subsequent embedding in Epon does provide optimal preservation of the structure, allowing semi-thin and ultra-thin electron microscopy (see Holstein et al. 1988). This technique allows clear identification of atypical germ cells in the case of testicular intraepithelial neoplasia (CIS/TIN), because of their nuclear structure and large amounts of intracytoplasmic glycogen granules (Fig..48b). However, this material is not suitable for the application of modern histological techniques such as immunohistochemistry or in situ hybridization, which provide evidence of gene expression at the protein and mrna levels. Therefore, fixation in Bouin s solution and embedding in paraffin wax is recommended (Figs..48c,.49a c). For histological evaluation, the biopsy sample should be about the size of a rice grain, showing about tubular cross-sections that are shown to be representative of the whole organ (Holstein et al. 1988). However, taking three biopsy samples on each side, our own data of score analysis revealed that there are regional differences within the same testis in respect of spermatogenesis, indicating focal impairment in about 12% of testis specimens (Fig..52b). In addition, the diagnostic safety in the detection of CIS is significantly increased by taking multiple testicular biopsy samples (Kliesch et al. 2003)..9.3 Evaluation Histological evaluation has first to detect and/or to exclude the presence of atypical germ cells (CIS/TIN), whichareknowntobetheprecursorsofmostseminomatous and nonseminomatous germ cell tumours, with the exception of spermatocytic seminoma (Dieckmann and Huland 2001). On paraffin sections these cells can be detected by immunohistochemistry using Histology Cryopreservation Fig..47. Scheme of testicular biopsy from three different locations per testis (modified according to Weidner et al. 2002) TESE

3 456 Diagnostic Tools nsp a b c Fig..48a c. Seminiferous tubules containing carcinoma in situ (CIS). a Formalin-fixed, paraffin section; haematoxylin and eosin staining. Note severeshrinkageartefactsofthetissue. b Glutaraldehyde fixation, semi-thin section; methylene blue staining. Note typical large nuclei including numerous nucleoli, and dark cytoplasmic glycogen (arrow)whichcanbeselectivelystained by PAS (arrow)(inset). c Bouin fixation, paraffin section, immunohistochemical staining against placenta-like alkaline phosphatase (PLAP). Note membrane-bound immunoreaction of CIS cells (arrow). (nsp Seminiferous epithelium showing intact spermatogenesis.) Primary magnification: a 20;b c 40 a b c Fig..49a c. Testicular histology. a Seminiferous tubules showing intact spermatogenesis. b Mixed atrophy showing maturation arrest at the level of early round spermatids (sda) or spermatogonia (sga), only Sertoli cells (SCO) or only lamina propria (tubular shadows = ts) in adjacent tubules. Note intratubular concentric spherical concrements derived from basal lamina (sk). c Prepubertal seminiferous cord within an adult testis showing undifferentiated Sertoli cells indicated by round to oval nuclear appearance compared to nuclei of normal Sertoli cells (inset = magnification of rectangle a). a c Paraffin sections, haematoxylin and eosin staining; primary magnification: a 40,b 20,c 40 different markers, and most commonly by the presence of placenta-like alkaline phosphatase (PLAP) (Fig..48c) (Beckstead 1983). Histological evaluation of any testicular tissue showing impaired spermatogenesis often provides so-called mixed atrophy (Sigg 1979), i.e. the simultaneous occurrence of seminiferous tubules showing at least qualitatively intact spermatogenesis, spermatogenic arrest at different levels of spermatogenesis including tubules with just Sertoli cells (Sertoli cell only = SCO) or even just lamina propria (tubular shadows) in adjacent tubules within the same testis (Fig..49a c).therefore, a semi-quantitative score count evaluation, i.e. according to Johnson (1970) or Bergmann and Kliesch

4 .9 Evaluation of Testicular Biopsy Samples from the Clinical Perspective 457 c a b d Fig..50a d. Histological evaluation using different methodological approaches. a Meiotic arrest as indicated by megalospermatocytes (arrow) and defects in spermiogenesis indicated by multinucleated spermatids (arrowheads). Semi-thin section, methylene blue, primary magnification: 40. b Double immunohistochemistry against s-phase-related Ki-67 protein staining spermatogonial nuclei (arrows), and vimentin intermediate filaments staining Sertoli cell cytoplasm (arrowheads). Paraffin section, haematoxylin counterstaining; primary magnification: 40. c, d In situ hybridization against protamine mrna expression in early round spermatids (arrows); c normal spermatogenesis; d hypospermatogenesis. Note the reduced number of labelled spermatids in the case of hypospermatogenesis despite normal histological appearance (arrowhead). Paraffin section, haematoxylin counterstaining; primary magnification: 40 (1998), is necessary. The Johnson score provides a scoring of every tubule within a given histological section, and is highly recommended for oligozoospermic patients, because a high correlation between testicular biopsy score and sperm count is found. However, oligozoospermia is not a current indication for testicular biopsy. In contrast, the score according to Bergmann and Kliesch (1998) (Fig..50a, b) is based only on the percentage of tubules within the biopsy section showing elongated spermatids, because the occurrence of these spermatids is of main interest in most cases when TESE and ICSI are performed. Histological evaluation additionally has to include a consideration of cytological alterations. Meiotic defects resulting in so-called megalospermatocytes (Holstein and Eckmann 1986; Johannisson et al. 2003) or multinucleated spermatids representing defects in spermiogenesis (see Holstein et al. 1988) can be recognized on semi-thin sections without any gene or protein expression analysis (Fig..51a). Estimation of spermatogonial mitotic activity, which is known to be reduced together with spermatogenic impairment, requires an immunohistochemical approach using antibodies against s-phase-related proteins such as K-67 or PCNA (Fig..51b) (Steger et al. 1998). In biopsy samples showing hypospermatogenesis, in situ hybridization revealed a reduced number of spermatids showing protamine gene expression (Fig..51c, d) (Steger et al. 2001) which was later confirmed by quantitative polymerase chain reaction (PCR) analysis (Steger et al. 2003). These data gave evidence that hypospermatogenesis results from different defects and impairments in germ cell development and differentiation. Alterations of somatic Sertoli cells are regularly found as signs of differentiation deficiency. This is suggested on routine paraffin sections by round to oval nuclei in Sertoli cells within immature seminiferous cords compared to the normal irregular shape with large and numerous clefts of Sertoli cells within normal seminiferous epithelium (Fig..49c), and was first proved by Bruning et al. (1993) using computer-assisted three-

5 458 Diagnostic Tools ts ts b ts ts a c d Fig..51a d. Immunohistochemistry of Sertoli cell differentiation. a, b Androgen receptor expression in Sertoli cell nuclei of normal seminiferous epithelium (a)and in prepubertal seminiferous cords (arrows). Note only weak expression in some Sertoli cellnuclei in prepubertal seminiferouscords. c Persistence of anti-müllerian hormone expression in Sertoli cells in prepubertal seminiferous cords. a cparaffin sections,haematoxylincounterstaining; primary magnification: 40. d Typical testicular histology of a Klinefelterpatient showing focal Leydig cell hyperplasia (arrow), and total atrophy of the seminiferous epithelium resulting in tubular shadows (ts = only lamina propria).paraffin section, haematoxylin and eosin staining;primarymagnification: a 10 dimensional reconstruction. Sertoli cell differentiation deficiency shown by different markers is now widely accepted to be associated with spermatogenic impairment, and was reviewed by Sharpe et al. (2003). Sertoli cells are the only cells within the seminiferous epithelium expressing androgen (Fig..51) as well as FSH receptors. In cryptorchidism, this androgen receptor expression is significantly reduced, as shown by Regadera et al. (2001) using quantitative immunohistochemistry. This is also true for Sertoli cells within premature seminiferous cords found in infertile men (Fig..51b), which additionally show the persistence of anti-müllerian hormone expression (Fig..51c) (Steger et al. 1996). Leydig cells regularly show hyperplasia, which is typically found associated with Sertoli cell only syndrome, i.e. in Klinefelter patients (Fig..51c). Taken together, score count evaluation together with a cytological analysis (Fig..52a, b) provide the opportunity for causal histological evaluation, giving rise to retrospective studies considering histological evaluation and successful TESE/ICSI. Such studies have the potential to facilitate successful assisted reproduction. Testicular biopsy is an invasive surgical operation withahighimpactonpatientswithseverespermatogenic impairment, and should therefore be performed only after considering strict criteria of indication, surgical procedure and histological evaluation in qualified centres that are certified, i.e. by the European Academy of Andrology (EAA).

6 .9 Evaluation of Testicular Biopsy Samples from the Clinical Perspective 459 Fig..52.a Form for evaluation

7 460 Diagnostic Tools Fig..52.b Example of biopsy evaluation of a hypergonadotrophic azoospermic patient showing total Sertoli cell only syndrome within the right and left testis, and additional focal areas of spermatogenesis within the upper and lower pole of the left testis. Cytoplasmic analysis also revealed an impairment of spermiogenesis (multinuclear spermatids)

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