The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.

/pdf/evaluation-of-chromatin-condensation-in-human-spermatozoa-a-4zshsz7o6w.pdf

Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining.

This study set out to establish a new method, using flow cytometry, to evaluate leukocytes in semen. Ejaculates of 59 males, asymptomatic for genitourinary infections, were examined. Routine semen analyses were carried out as well as peroxidase and polymorphonuclear granulocyte-elastase detection. Leukocytes were detected combining flow cytometry and monoclonal antibodies (anti-CD45, anti-CD53). This technique reliably assessed the total number of leukocytes and differentiated subpopulations even at low concentrations. The peroxidase test and elastase determination showed good specificity, but only moderate sensitivity versus flow cytometry combined with monoclonal antibodies. No significant association was observed between semen parameters and leukocytospermia whether evaluated by conventional methods or flow cytometry except for a moderate correlation between spermatozoa and CD53-positive cell concentrations. A first comparison of data from patients grouped on the basis of leukocytospermia (>10(6) white blood cells, WBC/ml) or non-leukocytospermia revealed no significant differences in semen parameters; lowering the threshold value for leukocytospermia to 2x10(5) WBC/ml, sperm concentration was reduced in the group with a low number of WBC identified by monoclonal antibodies. Flow cytometry using monoclonal antibodies was seen to be a simple, reproducible method that enables leukocytes in semen to be accurately detected and to identify WBC subpopulations without preliminary purification procedures.

/pdf/leukocyte-detection-in-human-semen-using-flow-cytometry-3zpw6m1x1b.pdf

Leukocyte detection in human semen using flow cytometry

Introduction
The exact causes of the decline in semen quality are not yet known, environmental factors have been considered to play an important role. Lead (Pb) and Cadmium (Cd) are two of the well-known reproductive toxicants to which humans are exposed occupationally and environmentally and can lead to negative effects on the testicular functions. The aim of this study was to evaluate lead and cadmium levels in seminal plasma of men with idiopathic oligoasthenozoospermia in comparison to fertile healthy controls and to correlate these levels with conventional semen parameters, sperm hypo-osmotic swelling (HOS) percentage, sperm DNA fragmentation percentage, and semen reactive oxygen species (ROS) levels.

/pdf/correlation-between-seminal-lead-and-cadmium-and-seminal-3ehjb9foxf.pdf

Correlation between seminal lead and cadmium and seminal parameters in idiopathic oligoasthenozoospermic males.

Sperm motion analysis was performed using computer-aided semen analysis (CASA) on nine normozoospermic human ejaculates following six fractionation schemes that included Percoll discontinuous gradient centrifugation, swim-up, hyper and hypo-osmotic stress in which 460013 400 tracks were analysed (depending on the scheme). In addition, the effect of ageing for 2 h in seminal plasma or re-addition of seminal plasma, on the kinematic properties was investigated. Analysis following sorting of the swimming velocity at 5 μm/sec intervals revealed the presence of two discrete normally distributed subpopulations selected on the basis of path velocity (VAP): these subpopulations were also present in the track speed (VCL) and progressive velocity (VSL) distributions. The validity of the two subpopulations was examined using a two-component normal mixture model and found to be highly statistically significant (p < 0.00001). The basis for the subpopulations was independent from the possession of midpiece vesicles or hyperactivation. Swim-up produced subpopulations of sperm that were significantly different from those obtained by Percoll separation. Compared with sperm in seminal plasma, swim-up and Percoll separation significantly increased the level of hyperactivated sperm at 2 h. Hyperactivation was inhibited by the re-addition of seminal plasma. Swim-up enhanced the proportion of sperm in the fast VAP subpopulation (independently of the level of hyperactivation) and whilst this altered distribution was maintained following the addition of homologous seminal plasma the mean velocity of the fast fraction was significantly decreased. Both swim-up and Percoll separation significantly increased the mean velocity of the fast subpopulation but the increase was greater with swim-up. This analysis highlights a way in which subpopulations may be detected and analysed. The procedure revealed fundamental differences in the kinematic properties of sperm processed by different methods that are used in assisted reproductive technology and showed that these were independent of hyperactivation.

Consistent presence of two normally distributed sperm subpopulations within normozoospermic human semen: a kinematic study

This chapter discusses the legal issues and the efforts that legislatures, courts, professional guidelines, programs, and patients have made to resolve them, in the hope of giving professionals some guidance in these evolving areas. Preserving male fertility through sperm collection and freezing is the most commonly recognized, and likely most frequently performed, of all fertility preservation measures. Donor gametes have also been involved with legal claims regarding embryo donation, discard, or custody of resulting children. Use of donor insemination to create a family raises additional legal issues for infertile men, their spouses or partners, and the sperm donors involved. Donor screening is required under a variety of state and federal laws, as well as professional guidelines. Posthumous reproduction raises additional legal complexities, including determination of the legal status of any resulting child under applicable law, which varies from jurisdiction to jurisdiction, within the United States as well as internationally.

Infertility in the Male: Semen white blood cell assay

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder ("marginal population"). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide.

Identification of two human sperm populations using flow and image cytometry.

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Identification of two human sperm populations using flow and image cytometry.