Spermatozoa isolation with Felix™ outperforms conventional density gradient centrifugation preparation in selecting cells with low DNA damage.

BACKGROUND: The optimization of spermatozoa preparation techniques in order to obtain cell fractions enriched with structurally and functionally "superior" spermatozoa is a key objective of the assisted reproduction industry. OBJECTIVES: The purpose of this study was to evaluate a recent development of an electrophoretic spermatozoa separation device (Felix™, Memphasys Ltd, Sydney, Australia) and to compare its performance with conventional spermatozoa preparation by density gradient centrifugation (DGC). Particular attention was paid to the evaluation of sperm DNA/nuclear integrity. MATERIALS & METHODS: A cohort of 29 human semen samples was studied. Semen samples were analyzed fresh and after DGC or Felix™ preparation. Semen parameters monitored included sample volume, sperm count, total motility, progressive motility, sperm DNA fragmentation using the Sperm Chromatin Structure Assay and sperm DNA oxidation. RESULTS: Spermatozoa preparation with Felix™ resulted in significantly improved spermatozoa fractions with higher progressive motility, lower sperm DNA fragmentation, and lower sperm DNA oxidation compared with raw semen and DGC-prepared spermatozoa. DISCUSSION & CONCLUSION: The data collected in this study support the preparation of spermatozoa by the Felix™ system as it allows selection of spermatozoa with the highest progressive motility as well as the lowest nuclear/DNA damage. These improved sperm parameters, along with the fact that the Felix™ separation process is very fast and highly standardized, should be of great interest to the assisted reproduction technologies industry.

Computer-aided sperm analysis, the new key player in routine sperm assessment.

For sperm analysis, important inter-laboratory variations have been observed in manual analyses. In this study, a computer-aided sperm analysis (CASA) system was assessed versus manual technique, and specific software modifications were operated to fit the David's classification already used in the laboratory. Four parameters were studied (concentration, motility, vitality and morphology), and at least 30 semen samples from 30 different patients have been tested. Manual and automated analyses were compared using a least-squares regression line analysis, Student's t test, Bland-Altman plots and Passing-Bablok regressions. Repeatability was also assessed, and coefficients of variation (CV) were calculated. Both manual and automated methods gave similar results for sperm concentration (n = 150), motility (n = 30), vitality (n = 90) and morphology (n = 90). Repeatability always showed a decrease in the CV with automated analysis; for example in normal range of sperm values, CV for manual and CASA analyses were, respectively, 9.0% versus 4.4% for sperm concentration, 5.2% versus 4.1% for motility, 7.3% versus 4.2% for vitality and 11.4% versus 4.1% for morphology. All parameters were comparable between automated and manual analysis, and repeatability measures confirm the more reliable values of the SCA compared to those of manual analysis.

Investigation of male infertility using quantitative comparative proteomics.

Male factors account for 40% of infertility cases. The identification of differentially expressed proteins on spermatozoa from fertile and infertile men can help in the elucidation of the molecular basis of male infertility. The aim of this study was to compare sperm proteomes from 3 different groups: fertile men, normozoospermic men consulting for infertility, and normozoospermic men with an impaired capacity for fertilization (IVF-failure). We used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and LC-MS analysis to identify proteins that are differentially expressed. A total of 348 unique proteins were identified and quantified. The analysis identified 33 proteins that were differentially expressed in the IVF-failure group vs the fertile group. Comparison of the infertile and fertile groups revealed that 18 proteins appeared to be differentially expressed. Four proteins were similarly altered in the IVF-failure and infertile groups: semenogelin 1 (SEMG1), prolactin-induced protein (PIP), glyceraldehyde-3-phosphate dehydrogenase (GAPDHS), and phosphoglycerate kinase 2 (PGK2). These protein markers were selected for validation using multiple reactions monitoring mass spectrometry (MRM-MS) and further confirmed by Western blot analysis. Overall, these results suggest that a panel of proteins may be used as biomarkers for future studies of infertility.

Electrophoretic characterization of the human sperm-specific enolase at different stages of maturation.

The presence of a sperm-specific enolase isoform (ENO-S) in human ejaculated spermatozoa was previously demonstrated. The objective of this study was to characterize this ENO-S in spermatozoa at different steps of maturation. Sperm ENO-S was characterized in testicular, epididymal, and ejaculated spermatozoa to determine whether any change occurred in the isoform patterns of this enzyme during epididymal maturation. In testicular sperm, ENO-S was present under 2 main bands named S1 and S3. In epididymal sperm, S1 and S3 bands and a prominent additional S2 band, with the same electrophoretic properties as the S isoform of ejaculated sperm, were visualized. In the testicular extracts obtained from testes in which no spermatozoa were visualized by histologic analysis, none of the 3 ENO-S bands was found. ENO-S exists as different isoforms (electrophoretic variants) in the different stages of sperm maturation. Passage through the epididymis seems to play a major role in the maturational process of this sperm-specific enolase.

Enolase isoforms activities in spermatozoa from men with normospermia and abnormospermia.

Total enolase as well as enolase-alphaalpha. (ENO-alphaalpha, ubiquitous) and enolase-S (ENO-S, sperm-specific) activities were measured in total and Percoll-selected sperm from 30 normospermic fertile men and 20 abnormospermic infertile patients. The total enolase activity was significantly higher in total sperm from patients with abnormospermia compared with normospermic patients (11.1 +/- 1.9 vs. 4.8 +/- 0.5 mlU/10(7) sperm P < .05). ENO-alphaalpha activity was significantly higher in total sperm from abnormospermic men than from normospermic men (P < .05). ENO-alphaalpha activity in Percoll-selected sperm was significantly lower compared with total sperm in both group of patients; however, for the same sperm fraction ENO-alphaalpha activity did not differ between normospermic and abnormospermic men. ENO-alphaalpha activity was related to the cell contamination ratio and to the percentage of spermatozoa with abnormal morphology. Furthermore, ENO-alphaalpha was positively correlated with the percentage of immature sperm showing an excess of residual cytoplasm. ENO-S activity was significantly higher in total sperm from normospermic patients than from abnormospermic patients (P < .05). ENO-S activity in Percoll-selected sperm was not significantly different compared with total sperm in both group of patients. However, this activity was significantly lower in Percoll-selected sperm from abnormospermic men compared with normospermic men (P < .05). ENO-S activity was not related to the cell contamination ratio but was significantly correlated with the percentage of spermatozoa with normal morphology. The 2 enolase isoforms seem to reflect 2 opposite aspects of sperm cells quality: ENO-alphaalpha is associated with abnormal spermatozoa, immature spermatozoa, or both; and ENO-S is associated with normal spermatozoa. As an additional index to distinguish normal from abnormal semen, the ENO-S:ENO-alphaalpha ratio was evaluated for total and Percoll-selected sperm in both groups. This ratio seems to be a new, valuable marker of the global sperm quality in a given semen sample, and may represent a predictive index of sperm fertilizing potential.

Sperm analysis by FISH in a case of t(17; 22) (q11; q12) balanced translocation: case report.

Individual sperm from men with balanced translocations have different chromosomal contents. Thus, an estimation of the overall sperm chromosomal imbalance of such patients could help to give the couple an adapted genetic counselling. We report here the study of a balanced translocation carrier, t(17;22) (q11;q12) whose reproductive history reported four miscarriages. Moreover, he had an abnormal semen analysis with oligoteratozoospermia. The meiotic segregation pattern was examined in 700 sperm, using fluorescence in-situ hybridization (FISH). Nineteen percent of the sperm had balanced translocations or were normal. All other sperm were unbalanced (81%) and their distribution was observed as follows: the frequencies of adjacent 1, adjacent 2 and 3:1 segregations were 12.9, 5.8 and 46.8% respectively. Among the segregations scored, 13.7% were related to second meiotic division abnormalities. Less than 2% of the total sperm scored were not explained. The 3:1 segregation was present at a very high rate, which is very unusual. In cases of balanced translocations, we believe that no general features can be drawn. Thus, the FISH technique may be very helpful for genetic counselling, which remains an important step and must be done with care.