WHO laboratory manual for the examination and processing of human semen

Oxidative stress occurs when the production of potentially destructive reactive oxygen species (ROS) exceeds the bodies own natural antioxidant defenses, resulting in cellular damage. Oxidative stress is a common pathology seen in approximately half of all infertile men. ROS, defined as including oxygen ions, free radicals and peroxides are generated by sperm and seminal leukocytes within semen and produce infertility by two key mechanisms. First, they damage the sperm membrane, decreasing sperm motility and its ability to fuse with the oocyte. Second, ROS can alter the sperm DNA, resulting in the passage of defective paternal DNA on to the conceptus. This review will provide an overview of oxidative biochemistry related to sperm health and will identify which men are most at risk of oxidative infertility. Finally, the review will outline methods available for diagnosing oxidative stress and the various treatments available.

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Oxidative stress and male infertility—a clinical perspective

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2002.tb02599.x

Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques.

Role of reactive oxygen species in male infertility.

Sperm DNA integrity is essential for the accurate transmission of genetic information. It has a highly compact and complex structure and is capable of decondensation-features that must be present in order for a spermatozoon to be considered fertile. Any form of sperm chromatin abnormalities or DNA damage may result in male infertility. In support of this conclusion, it was reported that in-vivo fecundity decreases progressively when > 30% of the spermatozoa are identified as having DNA damage. Several methods are used to assess sperm chromatin/DNA, which is considered an independent measure of sperm quality that may yield better diagnostic and prognostic approaches than standard sperm parameters (concentration, motility and morphology). The clinical significance of this assessment lies in its association not only with natural conception rates, but also with assisted reproduction success rates. Also, it has a serious impact on the offspring and is highly prognostic in the assessment of fertility in cancer patients. Therefore, screening for sperm DNA damage may provide useful information in cases of male idiopathic infertility and in those men pursuing assisted reproduction. Treatment should include methods for prevention of sperm DNA damage.

/pdf/role-of-sperm-chromatin-abnormalities-and-dna-damage-in-male-5a2riixlg7.pdf

Role of sperm chromatin abnormalities and DNA damage in male infertility

A method has been developed for quantifying the residual cytoplasm present in the midpiece of human spermatozoa, based upon the imaging of NADH oxidoreductase activity. This procedure used NADH and nitroblue tetrazolium as electron donor and acceptor, respectively, and resulted in the discrete staining of the entire midpiece area, including the residual cytoplasm. Image analysis techniques were then used to generate binary images of the midpiece, from which objective measurements of this cellular domain could be undertaken. Such data were found to be highly correlated with biochemical markers of the cytoplasmic space, such as creatine kinase (CK) and glucose-6-phosphate dehydrogenase (G-6-PDH), in sperm populations depleted of detectable leukocyte contamination. Morphometric analysis of the sperm midpiece was also found to reflect semen quality in that it predicted the proportion of the ejaculate that would be recovered from the high-density region of Percoll gradients and was negatively correlated with the movement and morphology of the spermatozoa in semen. Variation in the retention of excess residual cytoplasm was also associated with differences in the functional competence of washed sperm preparations, both within and between ejaculates. Thus, within-ejaculate comparisons of high- and low-density sperm subpopulations revealed a relative disruption of sperm function in the low-density fraction. This disruption was associated with the presence of excess residual cytoplasm in the midpiece, high concentrations of cytoplasmic enzymes, and the enhanced-generation reactive oxygen species (ROS). Functional differences between individual high-density Percoll preparations were also negatively correlated with the area of the midpiece and the corresponding capacity of the spermatozoa to generate ROS. These findings suggest that one of the factors involved in the etiology of defective sperm function is the incomplete extrusion of germ cell cytoplasm during spermiogenesis as a consequence of which the spermatozoa experience a loss of function associated with the induction of oxidative stress.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.1996.tb01783.x

Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa : Correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function

The addition of luminol to unprocessed semen samples resulted in the generation of chemiluminescent signals, the intensity of which was highly correlated with the level of leukocyte contamination. Despite the spontaneous oxidant-generating capacity of seminal leukocytes, no correlations were observed between leukocyte contamination and the fertility status of the subjects or any aspect of the semen profile, including the motility of the spermatozoa or their performance in a hyaluronate penetration assay. Luminol-dependent chemiluminescence and leukocyte contamination were also correlated in washed sperm suspensions prepared either by repeated centrifugation or on discontinuous Percoll gradients. However, in such sperm suspensions, the spontaneous generation of oxidants by contaminating leukocytes (> 2 x 10(4) leukocytes/ml) was invariably associated with a decreased capacity for movement. Moreover, causative associations between leukocyte contamination, reactive oxygen species generation, lipid peroxidation and impaired sperm motility were revealed by experiments involving the selective addition or removal of activated leukocytes. From these observations we can conclude that low concentrations of leukocytes are a common feature of the human ejaculate and can impair sperm function, particularly in the absence of seminal plasma. These findings have implications for our understanding of the importance of leukocytospermia in defining the fertility of human spermatozoa in vivo and in vitro.

Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma.

Protective effect of antioxidants on the impairment of sperm motility by activated polymorphonuclear leukocytes.

PROBLEM: To develop a methodology to determine a) the leukocytic contribution to reactive oxygen species generation by human sperm suspensions and b) the therapeutic value of removing these cellular contaminants.



METHODS: Leukocytes were removed with paramagnetic beads or colloidal ferrofluids coated with anti-CD45 antibody. The sperm suspensions were monitored for oxidant generation by chemiluminescence, leukocyte contamination by immunocytochemistry, and fertilizing potential using zona-free hamster oocytes.



RESULTS: Percoll®-prepared human sperm suspensions exhibited a competence for PMA-induced reactive oxygen species generation which was significantly correlated with leukocyte contamination. However, the purified spermatozoa remaining after paramagnetic bead treatment, also demonstrated an intrinsic capacity for PMA-responsive reactive oxygen species generation and, freed from the oxidative stress created by the leukocytes, exhibited a significantly enhanced capacity for sperm-oocyte fusion.



CONCLUSIONS: Although human spermatozoa can generate reactive oxygen species, sperm function is inhibited by the additional oxidative stress created by contaminating leukocytes. Removal of these cells with paramagnetic beads enhances fertilizing potential.

On the use of paramagnetic beads and ferrofluids to assess and eliminate the leukocytic contribution to oxygen radical generation by human sperm suspensions.

A comparative analysis has been undertaken of the behaviour of probes targeting the outer acrosomal membrane (Arachis hypogaea lectin) or constituents of the acrosomal vesicle (Pisum sativum lectin or monoclonal antibody CRB9) following the induction of the acrosome reaction in human spermatozoa with the ionophore A23187. The results obtained with these two classes of reagent were highly correlated (r = approximately 0.8), although the absolute rates of acrosome reaction were significantly different; the probe targeting the outer acrosomal membrane (A. hypogaea) consistently gave higher results than either of the probes targeting the acrosomal contents. Time-dependent studies, employing a double-labelling technique, indicated that this difference was apparent from the earliest time point examined (15 min after A23187 addition) and reflected the more rapid dissipation of the A. hypogaea lectin from the acrosomal region of the cell than either of the probes targeting the acrosomal vesicle. These results indicate that the outer acrosomal membrane is dispersed from acrosome-reacting human spermatozoa more rapidly than certain major constituents of the acrosomal vesicle and have possible implications for the design of diagnostic assays focusing on this aspect of human sperm function.

Andrology: Analysis of the ability of three probes targeting the outer acrosomal membrane or acrosomal contents to detect the acrosome reaction in human spermatozoa

J. Brindle

Papers

Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa : Correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function

Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma.

Protective effect of antioxidants on the impairment of sperm motility by activated polymorphonuclear leukocytes.

On the use of paramagnetic beads and ferrofluids to assess and eliminate the leukocytic contribution to oxygen radical generation by human sperm suspensions.

Andrology: Analysis of the ability of three probes targeting the outer acrosomal membrane or acrosomal contents to detect the acrosome reaction in human spermatozoa