Simultaneous expression analysis of deleted in azoospermia-family genes and CDC25A: their potential as a predictor for successful testicular sperm extraction

infections, varicocele, hypogonadotropic hypogonadism, chromosome abnormalities, and obstruction or agenesia of the seminal ducts. The study was designed in accordance with the Helsinki Declaration and its last modification (Tokyo 2004) on human experimentation, and it was approved by the Ethics Committees from Universidad Maimónides and the Centro de Estudios en Genética y Reproducción. Informed Consent was obtained from all patients. The diagnosis of azoospermia was established on the basis of the independent analysis of, at least, two semen samples collected 1 week apart. The serum concentrations of FSH (normal range: 1.5–7 mIU ml−1), LH (normal range: 1.1–9 mIU ml−1), and testosterone (normal range: 10–30 pmol ml−1) were measured and fell into the normal range in all patients. Azoospermic patients underwent a diagnostic testicular biopsy and sperm retrieval (TESE) by microsurgery and agreed to provide a small piece of testicular tissue (5 mm in diameter) for research purposes. The testicular histopathology was categorized according to the most advanced degree of spermatogenesis, and the biopsies were classified either as HS (n = 5) or MA (n = 3), according to McLachlan et al. (2007).10 DAB (3,3’-diaminobenzidine) immunohistochemistry was performed to localize DAZ family proteins and CDC25A in each biopsy. Negative controls were processed simultaneously by omitting the primary antibody or preabsorbing the primary antibody with specific synthetic peptides. Relative quantitation of gene expression by Real-time PCR of the DAZ gene family and CDC25A was calculated using standard curves and normalized to actin in each sample. Immunohistochemical analysis showed that in all biopsies with HS and MA, expression of DAZ was mainly detectable in the cytoplasm of spermatogonia clusters, near the basal lamina of the seminiferous tubules (Figure 1a), and DAZL was detectable in the cytoplasm of some spermatogonia and spermatocytes (Figure 1b). BOULE and its downstream substrate CDC25A shared a similar pattern of expression in germ cell cytoplasm (Figure 1c and 1d). Interestingly, in one biopsy diagnosed with MA, no immunoexpression of both proteins was detected, but we were able to detect BOULE mRNA in this particular biopsy, suggesting that the translation of BOULE might be regulated by another RNA-binding protein. We did not found an association between the expression levels of the DAZ family genes analyzed by real-time PCR with the diagnosis of Dear Editor, Infertility is a major health problem affecting 10%–15% of couples seeking to have children, and a male factor can be identified in about half of these cases.1 Nonobstructive azoospermia is one of the causes of male infertility (10%), resulting from testicular failure.2 The most common histological patterns in these patients are hypospermatogenesis (HS), maturation arrest (MA), and Sertoli cell-only syndrome (SCO).3 The search for sensitive and specific markers of spermatogenesis that could better predict the sperm retrieval rates in patients with nonobstructive azoospermia can lead to improved management of male infertility. DAZ (Deleted in Azoospermia) gene family has been extensively studied because the microdeletions containing DAZ genes in the Y chromosome are associated with a variety of testicular failures and impaired spermatogenesis.4–6 DAZ gene family consists of two autosomal genes, BOULE and DAZ-L (DAZ-like), and the DAZ gene cluster on chromosome Y. These genetic factors encode for RNA-binding proteins that are mainly expressed in germ cells and are considered essential for male fertility.5 Recently, the members of the cell cycle regulators CDC25 family were recognized as potential substrates for DAZ family proteins. Particularly, CDC25A is abundantly expressed in the testis and functions in the G1-S transition and M-phase exit, suggesting a role in mitotic or meiotic regulation of spermatogenesis.7–9 The analysis of single DAZ gene has shown that its dysfunction leads to abnormal spermatogenesis and may cause infertility. They were, however, poorly associated to sperm recovery during assisted reproductive treatments. Whether the simultaneous expression of the members of the DAZ gene family and its substrates may provide better information of testicular damage and success of sperm recovery in infertile patients has not been yet analyzed. We evaluated eight men (29–38-year-old) with idiopathic infertility and nonobstructive azoospermia diagnosed by open testicular biopsy. None of these patients showed genitourinary Simultaneous expression analysis of deleted in azoospermia‐family genes and CDC25A: their potential as a predictor for successful testicular sperm extraction