Testicular biopsy is a crucial assessment in reproductive practice with diagnostic and prognostic importance for assisted reproductive technologies (ARTs) and risk of testicular neoplasia. Endocrine and genetic tests cannot reliably distinguish obstructive azoospermia (OA) from non-obstructive azoospermia (NOA) or predict recovery of mature spermatids by testicular sperm extraction (TESE). Currently, divergent histological reporting systems and the use of imprecise terminology seriously degrade the value of the literature on TESE recovery rates and hamper evaluation of treatments and research on genotype-phenotype relationships. The rising incidence of testis cancer and carcinoma in situ (CIS), especially in infertile populations, requires that every effort be made for its early detection. We provide a systematic approach to the histological classification of spermatogenic disorders and detection of CIS in adult patients. We evaluate a large consecutive series of bilateral biopsies from infertile men and report (i) the frequency of bilateral or discordant patterns that supports the use of bilateral biopsy for comprehensive evaluation and (ii) a high prevalence of mixed patterns, particularly within the hypospermatogenesis classification, that helps account for reported success of TESE. We propose a new diagnosis code for testicular biopsies that addresses the needs of ART clinicians and allows data storage and retrieval of value in clinical practice and research.

Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly expressed in testicular CIS, including many never reported in testicular neoplasms. Expression was further verified by semiquantitative reverse transcription-PCR and in situ hybridization. Among the highest expressed genes were NANOG and POU5F1, and reverse transcription-PCR revealed possible changes in their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and 12, reported as unstable in cultured ESCs. The close similarity between CIS and ESCs explains the pluripotency of CIS. Moreover, the findings are consistent with an early prenatal origin of TGCTs and thus suggest that etiologic factors operating in utero are of primary importance for the incidence trends of TGCTs. Finally, some of the highly expressed genes identified in this study are promising candidates for new diagnostic markers for CIS and/or TGCTs.

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NANOG is a new marker for testicular CIS and germ cell tumours and the high level of NANOG along with OCT-4 are determinants of the stem cell-like pluripotency of the preinvasive CIS cell. Timing of NANOG down-regulation in fetal gonocytes suggests that NANOG may act as a regulatory factor up-stream to OCT-4.

Purpose: Transcription factor activator protein-2␥ (TFAP2C, AP-2␥) was reported previously in extraembryonic ectoderm and breast carcinomas but not in the testis. In our recent gene expression study we detected AP-2␥ in carcinoma in situ testis (CIS, or intratubular germ cell neoplasia), precursor of testicular germ cell tumors. In this study we aimed to investigate the expression pattern of AP-2␥ and to shed light on this factor in germ cell differentiation and the pathogenesis of germ cell neoplasia.Experimental Design: We analyzed expression pattern of AP-2␥ at the RNA and protein level in normal human tissues and a panel of tumors and tumor-derived cell lines. In the gonads, we established the ontogeny of expression of AP-2␥ in normal and dysgenetic samples. We also investigated the regulation of AP-2␥ by steroids and retinoic acid.Results: We detected abundant AP-2␥ in testicular CIS and in testicular germ cell tumors of young adults and confirmed differential expression of AP-2␥ in somatic tumors. We found that AP-2␥ expression was regulated by retinoic acid in an embryonal carcinoma cell line (NT2). The investigation of ontogeny of AP-2␥ protein expression in fetal gonads revealed that it was confined to oogonia/gonocytes and was down-regulated with germ cell differentiation. In some prepubertal intersex cases, AP-2␥ was detected outside of the normal window of expression, probably marking neoplastic transformation of germ cells.Conclusions: AP-2␥ is developmentally regulated and associated with the undifferentiated phenotype in germ cells. This transcription factor may be involved in self-renewal and survival of immature germ cells and tissue-specific stem cells. AP-2␥ is a novel marker of testicular CIS and CISderived tumors.