Mahmood Morshedi

Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa

OBJECTIVE To evaluate the effects of oxidative stress on DNA and plasma membrane integrity of human spermatozoa. DESIGN Prospective cohort study. SETTING University-based, tertiary-care infertility center. PATIENT(S) Men (n = 10) undergoing infertility investigation. INTERVENTION(S) Purified populations of sperm with high motility were separated using Percoll density gradients. Then, spermatozoa were incubated with 0, 10, 100, and 200 microM hydrogen peroxide (H(2)O(2)) under capacitating conditions. MAIN OUTCOME MEASURE(S) Motion parameters were assessed by computer analysis. Genomic integrity was examined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. Plasma membrane integrity was evaluated by the annexin V-binding assay, a measure of phosphatidylserine translocation. RESULT(S) Under basal conditions, there was a significant and negative relationship between sperm motility and the percentages of sperm with DNA fragmentation and membrane translocation of phosphatidylserine. After a 2-h incubation, there was a significant, dose-dependent effect of H(2)O(2) on motion parameters (decrease) and DNA fragmentation (increase). The percentage of annexin V(-) live (normal) cells declined significantly as the level of oxidative stress increased. Although the percentages of annexin V(+) live cells (sperm depicting translocation of phosphatidylserine) and necrotic cells increased at the highest H(2)O(2) levels, these changes were not significant. CONCLUSION(S) In vitro sperm incubation with H(2)O(2) induces DNA fragmentation in a dose-dependent fashion. The sublethal effects of oxidative stress on motion parameters were not significantly associated with membrane translocation of phosphatidylserine.

Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities

Sperm morphology evaluated by new, strict criteria is a good predictor of outcome in in vitro fertilization (IVF). This study aimed (1) to determine whether the fertilization rate of preovulatory oocytes in patients with abnormal morphology can be improved by increasing insemination concentration at the time of IVF and (2) to evaluate the pregnancy outcome in patients with abnormal sperm morphology. Three groups were studied: (1) normal morphology, (2) good prognosis pattern, and (3) poor prognosis pattern. All other sperm parameters were normal. Group 3 had a lower overall fertilization rate, lower pregnancy rate/cycle, and lower ongoing pregnancy rate/cycle. Groups 2 and 3 showed a higher miscarriage rate, although not significantly different from group 1. By increasing insemination concentration from 2- to 10-fold, the fertilization rate in group 3 increased from 14.5% to 62.6%. However, pregnancy outcome did not improve. We conclude that patients with severe sperm head abnormalities have a lower ability to establish successful pregnancies, even though fertilization may be achieved.

Fragmentation of DNA in morphologically normal human spermatozoa

OBJECTIVE To evaluate DNA fragmentation in spermatozoa with normal morphological appearance. DESIGN Prospective study. SETTING Academic tertiary center. PATIENT(S) Fertile, subfertile, and infertile men were studied. INTERVENTION(S) Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick-end labeling assay and morphology assessment by phase contrast in the swim-up fractions. MAIN OUTCOME MEASURE(S) Simultaneous assessment of the percentage of normally shaped sperm and DNA fragmentation. RESULT(S) No DNA fragmentation was found in spermatozoa with normal morphology in any of the samples from the fertile group. In only one sample from the subfertile group did we observed normally shaped sperm cells exhibiting DNA fragmentation. However, in all the samples from the infertile group, we observed normal spermatozoa with DNA fragmentation. Spermatozoa from this late group exhibited a high proportion of DNA damage. CONCLUSION(S) In infertile men with moderate and severe teratozoospermia, the spermatozoa with apparently normal morphology present in the motile fractions after swim-up may have DNA fragmentation.

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.

Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility

OBJECTIVE To assess the effect of pentoxifylline on human sperm functions that are crucial to fertilization. DESIGN Prospective, controlled study. SETTING Academic tertiary care institute. PATIENT(S) Healthy male sperm donors. INTERVENTION(S) The effects of pentoxifylline (3.6 mM) on hyperactivated motility, sperm binding to the zona pellucida, and sperm protein tyrosine phosphorylation were evaluated. MAIN OUTCOME MEASURE(S) Hyperactivated motility was assessed by computer-assisted motion analysis, and tight binding of sperm to homologous zonae pellucidae was examined using the hemizona assay. Sperm protein phosphorylation was evaluated using indirect immunofluorescence with an antibody to phosphotyrosine (PY20). RESULT(S) Pentoxifylline significantly stimulated hyperactivated motility at 1 hour and 4 hours; it also significantly increased sperm binding to the zona pellucida and enhanced sperm tail tyrosine phosphorylation at 4 hours under capacitating conditions. There was a statistically significant correlation between hyperactivated motility and sperm tail protein phosphorylation. CONCLUSION(S) Pentoxifylline stimulates sperm functions that are essential to achieving fertilization under in vitro conditions in sperm obtained from fertile men. The enhancement of hyperactivated motility is associated with the stimulation of sperm tail tyrosine phosphorylation, suggesting a causal relation and the involvement of a modulatory effect after cyclic adenosine monophosphate-dependent phosphorylation of intermediate proteins.

Presence and significance of somatic cell apoptosis markers in human ejaculated spermatozoa

Ejaculated spermatozoa, particularly in infertile men, have been shown to display morphological and biochemical features that are typical of an apoptotic phenotype in somatic cells. Deregulation of apoptosis is known to play roles in a number of disease processes, but roles for apoptosis in ejaculated spermatozoa and male infertility are poorly defined or have not been studied. Preliminary data demonstrated that populations of ejaculated spermatozoa express: (i) various degrees of plasma membrane translocation of phosphatidylserine and DNA fragmentation; and (ii) active caspase-3, the main executor of apoptosis in somatic cells, with an apparent exclusive cellular location to the mid-piece. Tests are currently being carried out on the effects of well-known apoptosis agonists and caspase inhibitors on such markers using purified populations of leukocyte-free ejaculated human spermatozoa. The main objective is to determine if somatic cell apoptosis markers are relevant indicators and/or causative factors of male infertility.

Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine

OBJECTIVE(S) [1] To evaluate sperm membrane damage during cryopreservation-thawing by the assessment of phosphatidylserine (PS) translocation and [2] to examine the relationship between reactive oxygen species (ROS) and cryopreservation-related alterations. DESIGN Prospective cohort study. SETTING University-based center. PATIENT(S) Men consulting for infertility and fertile donors (controls). INTERVENTION(S) Semen processing was performed by density gradient separation followed by cryopreservation and thawing. MAIN OUTCOME MEASURE(S) Membrane PS translocation was evaluated with annexin V binding, generation of ROS was detected by chemiluminescence, and motion parameters were assessed by computer analysis. RESULT(S) Annexin V binding was detected in the prefreeze fractions with high and low sperm motility. In the patient group, there were significantly higher postthaw levels of annexin V binding in both fractions when compared with prefreezing values. However, such induction of PS translocation was significantly higher in the fractions with high sperm motility. Significantly higher ROS levels were detected in prefreeze samples of the fractions with low sperm motility. CONCLUSION(S) In the population of men studied, [1] cryopreservation-thawing was associated with induction of membrane PS translocation; [2] postthaw ROS levels were lower than before freezing; and [3] neither annexin V binding results nor the generation of ROS were able to accurately predict sperm cryosurvival rates.

Plasma membrane integrity of cryopreserved human sperm: an investigation of the results of the hypoosmotic swelling test, the water test, and eosin-y staining

OBJECTIVE [1] To examine the relationship between sperm membrane integrity and motion parameters before and after cryopreservation; [2] to determine the capacity of the membrane integrity tests to predict the outcome of cryopreservation in fertile and infertile men; and [3] to examine the degree of agreement between tail and head membrane integrity of testicular and ejaculated immotile sperm cryopreserved for intracytoplasmic sperm injection. DESIGN Prospective study. SETTING Academic tertiary care institution. PATIENT(S) Fertile donors and normozoospermic oligozoospermic, and asthenozoospermic subfertile men. INTERVENTION(S) Semen samples were cryopreserved and thawed for analysis. MAIN OUTCOME MEASURE(S) Sperm membrane integrity and computer-assisted motion parameters. RESULT(S) The hypoosmotic swelling test and water test had a significant and positive correlation in the fresh and cryopreserved ejaculates of all groups. The results of the hypoosmotic swelling test correlated positively with the percent motility in the fresh ejaculates of fertile and subfertile men. None of the membrane integrity tests correlated with the cryosurvival rate in any group. In the ejaculated and testicular samples with no postcryopreservation motility, the simultaneous assessment of hypoosmotic swelling test and eosin showed that of 33% sperm exhibiting coiling with the hypoosmotic swelling test, only 9% were eosin negative, whereas 24% were eosin positive. CONCLUSION(S) [1] The water test may be a simpler replacement for the hypoosmotic swelling test; [2] none of the membrane integrity tests predicted sperm motility after cryopreservation; and [3] there was a high degree of disagreement between the hypoosmotic swelling test and eosin in the samples with no postcryopreservation motility.

Impact of Cryopreservation on Spermatozoa from Infertile Men: Implications for Artificial Insemination

The objective of this study was to evaluate the effect of cryopreservation-thawing on the quality of sperm from men with subfertile semen parameters. Twenty-seven patients with teratozoospermia, six of whom also had asthenozoospermia, were studied and their sperm parameters were compared to those of fertile donors (n = 71) in their fresh, post-thaw, and washed samples. After thawing, the percentage decrease in motility was significantly greater in patients than in donors, but the motility yield (post-thaw motility/prethaw motility) reached an average of 58% in the patient group vs. 68% in the donors (p = .02). No single characteristic of the fresh samples from patients or donors could significantly predict post-thaw outcome. For the patient group, however, multiple regression analysis provided a cutoff sperm concentration (50 x 10(6)/mL) and motility (40%) below which a very poor post-thaw recovery was obtained. The frozen-thawed-washed specimens had significantly higher velocity than the frozen-thawed samples, both in patients and donors. The results suggest that some patients with teratoasthenozoospermia yield acceptable sperm parameters after freezing-thawing-washing, and therefore these ejaculates could be used individually (or pooled) in artificial insemination.

Pentoxifylline stimulates various sperm motion parameters and cervical mucus penetrability in patients with asthenozoospermia

Summary. Pentoxifylline (PTX) was incubated in vitro with human spermatozoa to examine its effects on sperm motility characteristics and bovine cervical mucus penetrability (BCMP). Sperm motion parameters were assessed by computer‐assisted motion analysis (CASA) using HTM‐IVOS and BCMP was evaluated using the Penetrak kit. In vitro incubation with PTX (1 mg ml−1; 3.6 mm, 30 min) did not significantly change percentage motility, average path velocity (VAP), straight‐line velocity (VSL) or beat cross frequency (BCF) of spermatozoa from normozoospermic or asthenozoospermic samples. However, it significantly increased curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and hyperactivated motility (HA), and significantly decreased linearity (LIN) of spermatozoa from both samples. Pentoxifylline was found to increase BCMP scores for spermatozoa from asthenozoospermic samples, but did not affect scores for spermatozoa from normozoospermic samples. Bovine cervical mucus penetrability (BCMP) was found to be positively and significantly correlated with the percentage motility of both non‐PTX‐treated and PTX‐treated spermatozoa for asthenozoospermic samples. These results demonstrated that PTX enhanced several motion sperm parameters as well as BCMP in asthenozoospermic samples and suggest a potential use of the methylxanthine in infertile patients with motility defects undergoing artificial insemination.