The Significance ofLeukocyte Contamination

When it comes to the measurement of cellular ROS generation, human semen presents some significant problems that might not be encountered in other biological materials. Thus, one of the major drawbacks encountered in analyzing human semen from a ROS perspective is the presence of relatively high levels of leukocyte contamination. Every human semen sample is contaminated with leukocytes, the median concentration being around 24,900/mL or 91,000/ejaculate [18]. Although this may not seem like a large number of leukocytes, particularly given the concomitant presence of hundreds of millions of spermatozoa, it should be recognized that most of these contaminating cells (>80%) are granulocytes.

The dominance of granulocytes in the leukocytic profile is profoundly important because these cells are professional generators of ROS. Furthermore, at least some of the leukocytes contaminating human semen are in an activated state, generating a very high correlation coefficient (r = 0.8) between chemiluminescent measures of ROS generation and leukocyte concentration in semen (Fig. 14.1a, b) [18, 19]. Importantly, this correlation between leukocyte numbers and ROS generation extends across the entire range of leukocyte concentrations found in human semen (typically 104-105), far below the leukocyte concentration deemed as pathological by the World Health Organization [20] of 1 x 106/mL. Thus, even in semen samples that would not be classified as leukocytospermic by conventional criteria, the presence of leukocytes is still a significant source of ROS. Although the presence of a few thousand leukocytes may seem trivial, in terms of the measurement of ROS generation by spermatozoa it is of major importance because each leukocyte is log orders of magnitude more active than a spermatozoon in the generation of ROS. Even though some authors may assert that the semen samples subjected for analysis are not significantly contaminated with leukocytes from a clinical perspective (i.e., are not leukocytospermic), from a biochemical standpoint the presence of such contaminating cells is a major confounding factor in the measurement of ROS generation by spermatozoa that can render such determinations worthless.

The key to generating meaningful data when assessing ROS generation by entire sperm suspensions, as when chemiluminescence or amplex red are used, is to prepare the cells adequately. If unselected sperm populations are employed which have been prepared by repeated cycles of centrifugation and resuspension in a simple defined culture medium, the outcome is going to be a ROS signal that is dominated by the presence of contaminating leukocytes, dwarfing the relatively weak signal

Log Luminol in Semen (counts/10 sec)

Fig. 14.1 Leukocytes are a conspicuous feature of the free radical generating profile of human spermatozoa. (a) Immunocytochemical detection of leukocytes in human semen samples using a monoclonal antibody against the common leukocyte antigen (CD45). (b) In unfractionated semen samples, the ROS signal measured by luminol-dependent chemiluminescence is strongly correlated with leukocyte concentration measured using CD45 immunocytochemistry. Redrawn from Aitken et al. [18], by permission of Oxford University Press produced by the spermatozoa [21]. In order to affect the removal of contaminating leukocytes, some form of sperm fractionation procedure is required. This may take the form of a swim-up or swim-down procedure or, most commonly, discontinuous gradient centrifugation [21] . When sperm suspensions are subjected to such cell purification procedures, the ROS measurements obtained on these cell populations are significantly, and invariably, diminished (Fig. 14.2) [21, 22].

Very few authors have engaged the process of carefully dissecting the contribution made by leukocytes to the ROS signals generated by purified human sperm suspensions. However, where such studies have been conducted, the results are unequivocal in demonstrating that while leukocytes are the major source of ROS in such samples, defective spermatozoa are also capable of this activity [22, 23].

One of the earlier studies was conducted on unselected semen donors and examined ROS generation following fractionation of human semen samples on discontinuous Percoll gradients [22]. This study employed three variations of the luminol assay comprising: (1) luminol alone, (2) luminol plus horse radish peroxide to focus attention on H2O2 generation, and (3) luminol/peroxidase-dependent chemilumines-cence before and after the addition of catalase in order to secure a precise measure of extracellular H.O2 levels, as indicated in Fig. 14.2 . The results of this analysis again demonstrated that the low-density Percoll fractions exhibited significantly higher ROS signals than the purified high-density fractions, but that the latter still exhibited an impressive range of chemiluminescent responses [22, 23] (Fig. 14.2). Whether these signals were generated by spermatozoa, precursor germ cells, or residual leukocytes was then investigated using a monoclonal antibody against the common leukocyte antigen (CD45) to quantify the levels of white cell contamination. The results of one such analysis, illustrated in Fig. 14.3. indicated that the high-density Percoll fractions contained very few leukocytes, but whenever leukocytes were present a chemiluminescent signal was evident (Fig. 14.3). These samples also contained a low number of germ cells (0.1 x 104 per 107 spermatozoa), but the presence of these cells showed no relationship with the generation of ROS. As a result, when leukocyte concentrations are found to be low, much of the variation in ROS generation within these high-density Percoll fractions can be ascribed to the spermatozoa. Furthermore, these chemiluminescent signals were significantly higher in the spermatozoa of oligozoospermic patients than in normozoospermic controls (Fig. 14.3). An analysis focusing on high-density Percoll fractions from oligozoospermic samples, from which all detectable leukocyte contamination had been excluded, clearly demonstrated that the spermatozoa of these patients were significantly more active than controls in generating ROS [23].

Intriguingly, not only did the purified spermatozoa of oligozoospermic specimens generate higher levels of ROS, but the low-density fractions invariably contained significantly more leukocytes than normozoospermic control specimens (Fig. 14.3) [23]. This link between defective sperm function and leukocytic infiltration into the ejaculate has been observed subsequently [24, 25] and may represent a physiological leukocytic response to the presence of defective, moribund spermatozoa in the male reproductive tract, possibly triggered by apoptotic markers appearing on the surface of these cells [25].

Fig. 14.2 Comparison of the ROS-generating potential of spermatozoa recovered from the high and low-density regions of Percoll gradients. Purification of these cell populations in order to diminish the level of leukocyte contamination leads to a significant reduction in the ROS signal as measured by: (a) luminol; (b) luminol and peroxidase, and (c) the change in luminol and peroxidase chemiluminescence observed following the addition of catalase, in order to focus attention of extracellular H2O2. Boxes indicate 25th and 75th percentiles while the horizontal lines through the box represent the 50th percentile (median). Vertical lines indicate the 10th and 90th percentile limits of the data, while extreme results outside of these limits are represented as single data points. Redrawn from Aitken and West [22] by permission of John Wiley and Sons

Fig. 14.2 Comparison of the ROS-generating potential of spermatozoa recovered from the high and low-density regions of Percoll gradients. Purification of these cell populations in order to diminish the level of leukocyte contamination leads to a significant reduction in the ROS signal as measured by: (a) luminol; (b) luminol and peroxidase, and (c) the change in luminol and peroxidase chemiluminescence observed following the addition of catalase, in order to focus attention of extracellular H2O2. Boxes indicate 25th and 75th percentiles while the horizontal lines through the box represent the 50th percentile (median). Vertical lines indicate the 10th and 90th percentile limits of the data, while extreme results outside of these limits are represented as single data points. Redrawn from Aitken and West [22] by permission of John Wiley and Sons

Fig. 14.3 Relationship between leukocyte concentration and ROS signals measured following treatment of the spermatozoa with the 12-myristate, 13-acetate phorbol ester. (a) Cells from the Percoll/semen interface; (b) cells from the low-density/high-density Percoll interface and (c) samples recovered in the high-density Percoll pellet. Closed circles represent fertile donors while open circles represent oligozoospermic patients. Whenever leukocytes are present, significant ROS signals are recorded. More leukocytes are present in the oligozoospermic specimens, particularly in the low-density Percoll fractions. However, when samples were generated lacking detectable leukocyte contamination (as in c), it is clear that the spermatozoa exhibited a range of ROS-generating activity with the oligozoospermic specimens being particularly active. Redrawn from Aitken et al. [23], by permission from BioScientifica

Fig. 14.3 Relationship between leukocyte concentration and ROS signals measured following treatment of the spermatozoa with the 12-myristate, 13-acetate phorbol ester. (a) Cells from the Percoll/semen interface; (b) cells from the low-density/high-density Percoll interface and (c) samples recovered in the high-density Percoll pellet. Closed circles represent fertile donors while open circles represent oligozoospermic patients. Whenever leukocytes are present, significant ROS signals are recorded. More leukocytes are present in the oligozoospermic specimens, particularly in the low-density Percoll fractions. However, when samples were generated lacking detectable leukocyte contamination (as in c), it is clear that the spermatozoa exhibited a range of ROS-generating activity with the oligozoospermic specimens being particularly active. Redrawn from Aitken et al. [23], by permission from BioScientifica

Such data clearly suggest that the spermatozoa are a source of ROS in cases of defective sperm function; however, this contribution cannot be detected if leukocytes are present in the same cell suspension. Any assessments of ROS generation by human sperm suspensions that do not rigorously exclude a contribution from infiltrating leukocytes are therefore seriously compromised. There are two major ways in which this problem can be solved. First, if whole sperm populations are to be analyzed, then the only solution is to physically remove the leukocytes from the suspension. This can be done using Percoll gradient centrifugation or electropho-retic sperm isolation [26] to achieve the initial purification of the sperm suspension followed by a cell extraction procedure employing magnetic beads or ferrofluids coated in a monoclonal antibody targeting the common leukocyte antigen [ 27] . Using this technique, human sperm suspensions can be rapidly cleared of contaminating leukocytes allowing assessments of ROS generation by sperm suspensions that are entirely focused on the gametes.

Furthermore, the purity of the sperm suspensions generated using such cell purification techniques can be validated using simple leukocyte provocation assays employing either FMLP (formyl methionyl leucyl phenylalanine) or opsonized zymosan as the stimulant. The basic principle behind these assays is that following the addition of chemiluminescence reagents such as luminol/peroxidase, an agonist is added to the sperm suspension that can only trigger ROS generation by phagocytic leukocytes. FMLP was the first stimulant to be used for the detection of leukocytes in human sperm suspensions [28, 29], but more recently opsonized zymosan has been employed for the same purpose [26] . The signals elicited by these two probes are highly correlated, both with each other and with the size of the contaminating leukocyte population [29] .

If such techniques are not applied, there is a strong possibility that the responses recorded are more reflective of the level of leukocyte contamination than abnormal redox activity on the part of the spermatozoa. For example, the claim that NGF (nerve growth factor) will stimulate ROS generation in suspensions of human spermatozoa [30] is almost certainly a measure of the level of leukocyte contamination in these preparations [29] .

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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