M.S. Wosnitzer

Review of Azoospermia

Azoospermia is classified as obstructive azoospermia (OA) or non-obstructive azoospermia (NOA), each having very different etiologies and treatments. The etiology, diagnosis, and management of azoospermia were reviewed and relevant literature summarized. Differentiation between these two etiologies is of paramount importance and is contingent upon thorough history and physical examination and indicated laboratory/genetic testing. OA occurs secondary to obstruction of the male reproductive tract, and is diagnosed through a combination of history/physical examination, laboratory testing, genetics (CFTR for congenital OA), and imaging studies. NOA (which includes primary testicular failure and secondary testicular failure) is differentiated from OA by clinical assessment (testis consistency/volume), laboratory testing (FSH), and genetic testing (karyotype, Y chromosome microdeletion, or specific genetic testing for hypogonadotropic hypogonadism). For obstructive azoospermia, management includes microsurgical reconstruction when feasible using microsurgical vasovasostomy or vasoepididymostomy. Microsurgical epididymal sperm aspiration with in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) is utilized for those cases not amenable to reconstruction. NOA management includes medical management for congenital hypogonadotropic hypogonadism and microdissection testicular sperm extraction with IVF/ICSI for appropriate candidates based on laboratory/genetic testing. Overall, this important review provides an updated summary of the most recent available literature describing etiology, diagnosis, and management of azoospermia.

Possible germ cell-Sertoli cell interactions are critical for establishing appropriate expression levels for the Sertoli cell-specific MicroRNA, miR-202-5p, in human testis

BackgroundTo examine human microRNA expression in fertile men and subsequently to compare expression patterns of miRNAs in fertile and infertile men, specifically men with Sertoli Cell Only (SCO) histopathology.MethodsTesticular tissues from men with azoospermia and SCO, as well as those of men with normal spermatogenesis, were analyzed. MicroRNA was isolated using the miRCURY™ RNA Purification Kit. A miRCURY LNA™ Universal RT system was used for detection of microRNA by quantitative real-time PCR. MicroRNA localization was performed by in situ hybridizations (ISH) on formalin-fixed paraffin embedded (FFPE) tissue utilizing miRCURY LNA™ microRNA ISH technology. Statistical analysis was performed by GenEx V5.0.ResultsMicroRNA expression was determined for 13 normal fertile men and 5 men with the confirmed diagnosis of diffuse SCO. MiR-202-5p expression was reduced by 17-fold (P < 0.00001) in tissue from SCO men compared to normal. MiR-34c-5p was reduced by 346-fold (P < 0.00001), miR-10b was reduced 18-fold (P < 0.00001), miR-191 was reduced 20-fold (P = 0.001) and miR-126 was reduced 40-fold (P < 0.00001)) in tissues from SCO compared to normal fertile men. Using ISH, miR-202-5p was localized to Sertoli cells of men with normal spermatogenesis, but not in the Sertoli cells of men with SCO.ConclusionNumber of miRNAs are differentially expressed in normal fertile men compared to men with SCO. MicroRNA-202-5p is localized to Sertoli cells and its expression dramatically differs between fertile men and men whose germ cells are depleted, suggesting a novel interaction for regulating microRNA expression between the somatic and germ cell components of the seminiferous epithelium.AbstractObjectifsEvaluer l’expression des microARN chez des hommes féconds puis comparer les profils d’expression de ces miRNAs chez des hommes féconds et des inféconds qui présentent plus particulièrement un syndrome de Sertoli seules (SCO) à l’histologie testiculaire.Matériel et MéthodesOnt été analysés des tissues testiculaires d’hommes avec azoospermie et SCO ainsi que ceux d’hommes avec spermatogenèse normale. Les miRNAs ont été isolés avec la trousse de Purification miRCURY™ RNA. Le système miRCURY LNA™ Universal RT a été utilisé pour la détection quantitative de miARNs par PCR en temps réel. La localisation des miARNs a été réalisée par hybridation in situ (HIS) sur des tissus fixés au formol et inclus en paraffine en utilisant la technologie miRCURY LNA™ microRNA ISH. Les analyses statistiques ont été faites avec GenEx V5.0.RésultatsL’expression des microARNs a été faite chez 13 hommes féconds et 5 hommes avec un diagnostic confirmé de SCO diffus. L’expression de miR-202-5p est réduite d’un facteur 17 (P < 0.00001) dans le tissu des hommes SCO par rapport au tissu des hommes à spermatogenèse normale. L’expression de miR-34c-5p est réduite d’un facteur 346 (P < 0.00001), celle de miR-10b d’un facteur 18 (P < 0.00001), celle de miR-191 d’un facteur 20 (P = 0.001) et celle de miR-126 d’un facteur 40 (P < 0.00001) dans les tissus des hommes SCO comparés à ceux des hommes à spermatogenèse normale. MiR-202-5p a été localisé par HIS dans les cellules de Sertoli des hommes à spermatogenèse normale, mais pas dans les cellules de Sertoli des hommes SCO.ConclusionsNombre de miARNs sont exprimés différentiellement chez les hommes féconds par rapport aux hommes SCO. MicroARN-202-5p est localisé dans les cellules de Sertoli et son expression diffère de façon marquée entre les hommes féconds et ceux dont les cellules germinales sont absentes; ceci suggère une nouvelle interaction – entre les cellules somatiques et germinales constitutives de l’épithélium séminifère – impliquée dans la régulation de l’expression des microARNs.

Varicocele and Hypogonadism

Accumulating evidence suggests that varicocele, long associated with male infertility, is also a risk factor for low testosterone levels. The exact pathophysiology of the negative effects of varicocele on testicular function is not well understood, but theories include venous stasis, increased testicular temperature, oxidative stress, and resulting toxic environment. While prior studies report conflicting effects of non-microsurgical varicocelectomy on testosterone level, recent literature demonstrates that microsurgical varicocelectomy improves testosterone levels in men with varicocele and low testosterone preoperatively.

Genetic evaluation of male infertility

Men with severe oligospermia (<5 million sperm/mL ejaculate fluid) or azoospermia should receive genetic testing to clarify etiology of male infertility prior to treatment. Categorization by obstructive azoospermia (OA) or non-obstructive azoospermia (NOA) is critical since genetic testing differs for the former with normal testicular function, testicular volume (~20 mL), and follicle-stimulating hormone (FSH) (1-8 IU/mL) when compared to the latter with small, soft testes and increased FSH. History and physician examination along with laboratory testing (following appropriate genetic counseling) is critical to accurate selection of genetic testing appropriate for azoospermia due to primary testicular failure as compared with congenital hypogonadotropic hypogonadism (HH). Genetic testing options include cystic fibrosis transmembrane conductance regulator (CFTR) testing for men with congenital absence of the vas, while karyotype, Y chromosome microdeletions (YCMD), and other specific genetic tests may be warranted depending on the clinical context of severe oligospermia or NOA. The results of genetic testing guide management options. The most recent techniques for genetic analysis, including sperm microRNA (miRNA) and epigenetics, are forming the foundation for future genetic diagnosis and therapeutic targets in male infertility.

Ubiquitin Specific Protease 26 (USP26) Expression Analysis in Human Testicular and Extragonadal Tissues Indicates Diverse Action of USP26 in Cell Differentiation and Tumorigenesis

Ubiquitin specific protease 26 (USP26), a deubiquitinating enzyme, is highly expressed early during murine spermatogenesis, in round spermatids, and at the blood-testis barrier. USP26 has also been recognized as a regulator of androgen receptor (AR) hormone-induced action involved in spermatogenesis and steroid production in in vitro studies. Prior mutation screening of USP26 demonstrated an association with human male infertility and low testosterone production, but protein localization and expression in the human testis has not been characterized previously. USP26 expression analysis of mRNA and protein was completed using murine and human testis tissue and human tissue arrays. USP26 and AR mRNA levels in human testis were quantitated using multiplex qRT-PCR. Immunofluorescence colocalization studies were performed with formalin-fixed/paraffin-embedded and frozen tissues using primary and secondary antibodies to detect USP26 and AR protein expression. Human microarray dot blots were used to identify protein expression in extra-gonadal tissues. For the first time, expression of USP26 and colocalization of USP26 with androgen receptor in human testis has been confirmed predominantly in Leydig cell nuclei, with less in Leydig cell cytoplasm, spermatogonia, primary spermatocytes, round spermatids, and Sertoli cells. USP26 likely affects regulatory proteins of early spermatogenesis, including androgen receptor with additional activity in round spermatids. This X-linked gene is not testis-specific, with USP26 mRNA and protein expression identified in multiple other human organ tissues (benign and malignant) including androgen-dependent tissues such as breast (myoepithelial cells and secretory luminal cells) and thyroid tissue (follicular cells). USP26/AR expression and interaction in spermatogenesis and androgen-dependent cancer warrants additional study and may prove useful in diagnosis and management of male infertility.

Bulbocavernosus muscle area measurement: a novel method to assess androgenic activity

Serum testosterone does not correlate with androgen tissue activity, and it is critical to optimize tools to evaluate such activity in males. Ultrasound measurement of bulbocavernosus muscle (BCM) was used to assess the relationship between the number of CAG repeats (CAGn) in the androgen receptor (AR) and the BCM size; the changes in the number of CAGn over age were also evaluated. Transperineal ultrasound measurement of the BCM was also performed. AR CAGn were determined by high performance liquid chromatography, and morning hormone levels were determined using immunoassays. Forty-eight men had CAG repeat analysis. Twenty-five were <30 years of age, mean 23.7 years (s.d. = 3.24) and 23 were >45 years of age, mean 53 years (s.d. = 5.58). The median CAGn was 21 (13–29). BCM area was greater when the number of CAGn were <18 as compared to the number of CAGn >24 (P = 0.04). There was a linear correlation between the number of CAGn and the BCM area R2 = 16% (P = 0.01). In the 45 to 65-years-old group, a much stronger negative correlation (R2 = 29%, P = 0.01) was noticed. In the 19 to 29-years-old group, no such correlation was found (R2 = 4%, P = 0.36). In older men, the number of CAGn increased with age (R2 = 32%, P = 0.01). The number of CAGn in the AR correlates with the area of the BCM. Ultrasound assessment of the BCM is an effective surrogate to evaluate end-organ activity of androgens. The number of CAGn may increase with age.