Complete globozoospermia associated with PLCζ deficiency treated with calcium ionophore and ICSI results in pregnancy.

Globozoospermia is an infrequent pathology in which spermatozoa lack acrosomes. Patients are considered sterile without IVF augmented with intracytoplasmic sperm injection (ICSI), as fertilization is impaired due to absence of oocyte activation. As far as is known, this is the first study to report results of a comprehensive approach to the treatment of the semen parameters, sperm DNA fragmentation, aneuploidy, transmission electron microscopy, Western blotting and immunofluorescence for detection of phospholipase C zeta (PLCzeta), as well as ICSI outcome, of an affected patient. Morphological evaluation and transmission electron microscopy revealed complete globozoospermia with significant duplicate heads and tails. Analysis for DNA damage revealed fragmentation rates of approximately 80% in semen and 15-23% in swim-up fractions. PLCzeta was not detected by immunofluorescence or Western blotting. Aneuploidy rates were within normal ranges. ICSI followed by oocyte activation with calcium ionophore resulted in high rates of fertilization, and an ongoing pregnancy was established after transfer of cryopreserved-thawed embryos.

Somatic cell apoptosis markers and pathways in human ejaculated sperm: potential utility as indicators of sperm quality.

In this study we extended earlier work to determine whether sperm respond to somatic cell apoptotic stimuli and whether apoptotic phenotypes are significant indicators of human sperm quality. We evaluated ejaculated sperm from fertile donors and subfertile patients following purification of fractions of high and low motility. In unstimulated conditions, caspase enzymatic activity was higher in motile fractions from subfertile patients than in donors, and was higher in low motility fractions from both groups. Staurosporine, but not a Fas ligand or H2O2, significantly increased caspase activity, but only in high motility fractions. Procaspase-3, -7 and -9 and low levels of active caspase-3, -7 and -9 were identified by immunoblot analysis. Apoptosis-inducing factor (AIF) was present in all samples but poly ADP-ribose polymerase-1 (PARP-1) was not detected. Phosphatidylserine translocation was significantly increased only with H2O2 treatment. In ejaculates of both subfertile and fertile men, we demonstrated the presence and activation of several proteins that are key constituents of apoptosis-related pathways in somatic cells, which may serve as markers for sperm quality.

Cryopreservation-Thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation.

The objective of these studies was to evaluate the effect of cryopreservation-thawing of human spermatozoa on DNA fragmentation and membrane integrity. This was a prospective, controlled cohort study, performed at a university-based infertility center. Ejaculates were examined from 5 donors and 16 men undergoing infertility evaluation. Purified sperm populations were prepared by gradient centrifugation, cryopreserved using a manual method and TEST-yolk buffer and glycerol (TYB-G), followed by quick-thaw. Annexin V binding was used for assessing membrane translocation of phosphatidylserine, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. The results were as follows: the percentage of live cells with intact membranes (annexin V-, live) was significantly reduced after cryopreservation-thawing. On the other hand, the percentages of live cells with phosphatidylserine translocation (annexin V-, live) and of necrotic (dead) cells increased significantly after thawing. TUNEL revealed percentages of cells with DNA fragmentation in the prefreeze and postthaw samples that were not significantly different. In a further attempt to examine differences in response to various cryoprotection protocols, experiments were carried out using no cryoprotection, glycerol alone, or TYB-G. Samples frozen with TYB-G demonstrated significantly higher percentages of live cells without phosphatidylserine translocation than the other conditions. We concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.