STUDY QUESTIONAre exosomal microRNAs (miRNAs) in seminal plasma (SP) useful as markers of the origin of azoospermia and the presence of sperm in the testis?SUMMARY ANSWEROur study demonstrated the potential of several miRNAs contained in small extracellular vesicles (sEVs) of seminal fluid as sensitive and specific biomarkers for selecting those azoospermic individuals with real chances of obtaining spermatozoa from the testicular biopsy.WHAT IS KNOWN ALREADYThere are no precise non-invasive diagnostic methods for classifying the origin of the sperm defects in semen and the spermatogenic reserve of the testis in those infertile men with a total absence of sperm in the ejaculate (azoospermia). The diagnosis of such individuals is often based on the practice of biopsies. In this context it is reasonable to study the presence of organ-specific markers in human semen that contains fluid from the testis and the male reproductive glands, which could help in the diagnosis and prognosis of male infertility. Additionally, seminal fluid contains high concentrations of sEVs that are morphologically and molecularly consistent with exosomes, which originate from multiple cellular sources in the male reproductive tract.STUDY DESIGN, SIZE, DURATIONA case and control prospective study was performed. This study compares the miRNA content of exosomes in semen samples obtained from nine normozoospermic fertile individuals (control group), 14 infertile men diagnosed with azoospermia due to spermatogenic failure, and 13 individuals with obstructive azoospermia and conserved spermatogenesis. Additionally, three severe oligozoospermic individuals (<5 × 106 sperm/ml) were included in the study.PARTICIPANTS/MATERIALS, SETTING, METHODSA differential high-throughput miRNA profiling analysis using miRNA quantitative PCR panels was performed in SP exosomes from azoospermic patients and fertile individuals.MAIN RESULTS AND THE ROLE OF CHANCEA total of 623 miRNAs were included in the miRNA profiling stage of the study. A total of 397 miRNAs (63.7%) were consistently detected in samples from all groups and statistically analysed, which revealed altered patterns of miRNA expression in infertile patients. We focused on the miRNAs that were differentially expressed between azoospermia as a result of an obstruction in the genital tract (i.e. having conserved spermatogenesis) and azoospermia caused by spermatogenic failure, and described, in a miRNA validation stage of the study, the expression values of one miRNA (miR-31-5p) in exosomes from semen as a predictive biomarker test for the origin of azoospermia with high sensitivity and specificity (>90%). The efficacy of the predictive test was even better when the blood FSH values were included in the analysis. Furthermore a model that included miR-539-5p and miR-941 expression values is also described as being useful for predicting the presence of residual spermatogenesis in individuals with severe spermatogenic disorders with diagnostic accuracy.LIMITATIONS, REASONS FOR CAUTIONFurther studi...
Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. We have studied the molecular pathology of SMA in 745 unrelated Spanish patients using PCR-RFLP, SMN gene dosage analysis, linkage studies, long-range PCR and direct sequencing. Our systematic approach allowed us to complete genetic testing and risk assessment in 736 SMA patients (98.8%). Females were more frequently affected by the acute form of the disease (type I), whereas chronic forms (type II-III) predominated in males (p<0.008). Absence of the SMN1 gene was detected in 671 patients (90%), and hybrid SMN1-SMN2 genes were observed in 37 cases (5%). Furthermore, we detected 13 small mutations in 28 patients (3.8%), four of which were previously identified in other populations (c.91dupT; c.770_780dup11; p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date only in Spanish patients (c.399_402delAGAG, p.Ile116Phe, p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG mutation accounted for 1.9% of all Spanish SMA patients. Finally, we discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and p.Met263Thr. Our results confirm that most SMA cases are due to large genetic rearrangements in the repetitive region of the SMA locus, resulting in absence-dysfunction of the SMN1 gene. By contrast, ancestrally inherited small mutations are responsible for only a small number of cases. Four prevalent changes in exons 3 and 6 (c.399_402delAGAG; c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70% of our patients with these subtle mutations. An SMN-SMN dimer model featuring tight hydrophobic-aromatic interactions is proposed to explain the impact of mutations at the C-terminal end of the protein.
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