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.

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.

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. K E Y W O R D Scomputer-aided sperm analysis, manual sperm analysis, sperm analysis

In this work, we examined whether spermatozoa (spz) from normospermic fertile patients and selected by a swim-up (S-U) procedure had a particular membrane fluidity related to their maturity and their lipid content as compared with the sperm cells from the whole ejaculate (total sperm). Swim-up selected sperm had a reduced cytoplasmic space as revealed by a lower creatine kinase (CK) activity compared with total sperm (2 +/- 1 vs. 12 +/- 5 mUI/10(7) spz, p < 0.05). The cholesterol (Chol) and total phospholipid (PL) contents were significantly lower in S-U selected sperm than in total sperm (0.72 +/- 0.08 vs. 1.20 +/- 0.30 nmol/10(6) spz for Chol and 1.77 +/- 0.17 vs. 2.78 +/- 0.50 nmol/10(6) spz for PL, p < 0.05) and such a decrease was observed for the three major membrane PL: phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM). However, these decreases were not associated with a change in either Chol/PL or PC/(PC + PE) molar ratios. Membrane fluidity estimated by fluorescence polarization remained comparable between the S-U sperm fraction and total sperm (fluorescence polarization anisotropy, r, which is inversely proportional to the fluidity: 0.235 +/- 0.006 vs. 0.230 +/- 0.005). The sperm membrane fluidity obtained in normospermic patients was compared with abnormospermic ones (oligoasthenoteratospermia). In abnormospermic patients, the membrane fluidity was decreased in migrated spermatozoa compared with total sperm (anisotropy: 0.210 +/- 0.010 vs. 0.250 +/- 0.013, p < 0.01). Our data suggest that the S-U method selected a subpopulation of mature spermatozoa characterised by a low content of Chol and PL, likely related to a reduced membrane area. The fact that Chol/PL and PC/(PC + PE) molar ratios were unchanged shows a maintenance of the membrane quality. This was confirmed by the fluorescence anisotropy measurement showing no difference in plasma membrane fluidity between S-U selected sperm and total sperm. In abnormal semen the migrated spermatozoa had a lower fluidity compared with total sperm suggesting a defective sperm function. These results bring new elements characterizing the S-U selected spermatozoa.

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