Two halves of a whole: Bilobed testis case report and implications in management of a rare condition.

Bilobed testicle is an exceedingly rare congenital malformation with only seven cases reported in the literature. We describe the case of a 39-year-old man who presented with a left-bilobed testicle, resembling testicular malignancy. Despite its rarity, bilobed testicle should be considered in the differential diagnosis when a testicular mass is detected. Once malignancy has sufficiently been ruled out, bilobed testicle is typically managed conservatively.

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.

BACKGROUND: The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men. METHODS: We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls. RESULTS: We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression. CONCLUSIONS: In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).

High quality RNA in semen and sperm: isolation, analysis and potential application in clinical testing.

PURPOSE: Male infertility is a complex health condition. To our knowledge there are no molecular biomarkers of male infertility. Sperm RNA is a potential biomarker for detecting sperm abnormalities and viability at infertility clinics. However, RNA use is hindered by its inconsistent quantity, quality, multiple cell types in semen and condensed sperm structure. MATERIALS AND METHODS: We tested the usefulness of high quality RNA isolated from mature sperm and whole semen by our protocol, which reduces RNA degradation by maintaining semen and protocol components at 37 C and decreasing processing time. We isolated RNA from 83 whole semen samples, 18 samples of motile sperm prepared by the swim-up protocol and 18 of sperm prepared by the standard Percoll gradient method. RESULTS: Electrophoretic and spectral analysis of RNA revealed high quality 18S and 28S rRNAs in 71 of 83 whole semen samples (86%) and 15 of 18 mature sperm swim-up samples (83%). However, high quality RNA was isolated from only 7 of 18 Percoll gradient sperm samples (39%). Interestingly quantitative reverse transcriptase-polymerase chain reaction analysis of 4 somatic and 10 germ cell markers showed that whole semen and swim-up samples had similar RNA profiles. RNA sequencing revealed that most encoded proteins were involved in mature sperm function, regulation of DNA replication, transcription, translation, cell cycle and embryo development. CONCLUSIONS: We believe that semen and sperm specific RNAs are highly informative biomarkers for germ cell stages and somatic cell contribution. Therefore, these RNAs could be valuable diagnostic indicators of sperm survival, fertilization and early embryogenesis, and could serve as a predictor of the in vitro fertilization prognosis.

Testicular touch preparation cytology in the evaluation of male infertility.

BACKGROUND: Male infertility is traditionally evaluated by tissue core biopsies of the testes. Touch preparations (TP) of these biopsies have been infrequently used. The aim of this study is to report our experience with using testicular biopsy TP for the evaluation of male infertility. MATERIALS AND METHODS: A retrospective search was performed for cases of testes biopsies with concurrent TP. These cases were evaluated for clinical information, specimen adequacy, and cytological-histological correlation. RESULTS: A total of 39 cases were identified from men with a mean age of 34 years (range 23 to 50 years). TP slides were satisfactory for evaluation in 31 (89%) cases, and less than optimal in four due to low cellularity, obscuring blood or air drying artifact. Cytopathology showed concordance with the biopsy in almost all cases. In one discordant case where the biopsies showed no active spermatogenesis, a rare sperm were identified on the TP. CONCLUSIONS: TP of the testis is a helpful adjunct to biopsy because of its ability to clearly evaluate all stages of spermatogenesis. These data demonstrate that TP cytopathology of the testes in our experience has an excellent correlation with both normal testicular biopsies and those showing pathological spermatogenesis, and in rare cases may provide added benefit in evaluating the presence of spermatogenesis for male infertility. Albeit uncommon, cytopathologists may be required to identify and evaluate spermatogenic elements in cytology specimens being submitted from men with infertility.