The use of methylxanthine derivatives such as pentoxifylline for the stimulation of sperm functions, especially motility, is well known. Pentoxifylline is a nonspecific inhibitor of phosphodiesterase that has stimulatory effects on sperm motility and motion characteristics like sperm velocity or hyperactiv-ity. The stimulatory effect is attributed to increased intracellular levels of cAMP via inhibition of its breakdown by cAMP phosphodiesterase. Pentoxifylline is also reported to enhance the acrosome reaction (32) presumably due to the increasing levels of cAMP. The results of pentoxifylline treatment in assisted reproduction are equivocal. Depending on the conditions, especially the time of stimulation relative to the capacitative state of the spermatozoa and the concentration of pentoxifylline in the medium, overstimulation can result in a premature acrosome reaction (12). Thus, pentoxifylline tends to be used on a limited basis in IVF programs and some programs choose to use pentoxifylline only in the preparation of epididymal and testicular sperm for assisted IVF.
Spermatozoa retrieved from the testis have not experienced the maturation-inducing influence(s) afforded during epididymal transport, and therefore, are in a different physiologic state than epididymal or ejaculated spermatozoa. Treatment of immotile or very poorly motile fresh or cryopreserved testicular spermatozoa with pentoxifylline very frequently simulates some form of motion, whether it is twitching, nonprogressive motility, or progressive motility. The goal in any ICSI procedure is to use spermatozoa that are viable, and motion is the best indicator ensuring both a functional (protective) plasma membrane and patent metabolic processes. The combination of these two attributes lends greater assurance that the DNA has not been made more vulnerable to the deleterious effects of ROS.
Platelet-activating factor (PAF) is a biologically active phospholipid thought to be a cellular mediator in reproduction that has been found in spermatozoa of many different species, including human (33). PAF has been reported to have positive effects on motility, capacitation, acrosome reaction, and oocyte penetration (34,35), and these stimulatory actions on sperm function can be inhibited by PAF antagonists (36). Although the molecular mechanism of action has yet to be fully elucidated, the positive effect of PAF on sperm function has led to its use in assisted reproduction. Roudebush et al. (37) reported that pregnancy rates in IUI cycles were significantly increased after the spermatozoa from normozoospermic males were prepared with a medium containing PAF.
Sperm motility is an important indicator of viability, especially when performing ICSI. In the absence of native or stimulated sperm motility, the assessment of viability becomes critical. There is a simple vitality test based on the semi-permeability of the intact and physiologically functional plasma membrane which causes spermatozoa to swell under hypo-osmotic conditions, when an influx of water results in an expansion of cell volume (6). This vitality test is known as the HOS test.
The HOS test can be used for specimens where the spermatozoa are all immotile. When setting up a dish for the ICSI procedure, a small (5 mL) drop of HOS solution is placed near the PVP drop and two extra drops of culture medium are placed nearby. A small volume of sperm suspension is placed in one of the extra drops. When spermatozoa are located, they are picked up in the ICSI micropipette and placed in the HOS solution. Immediately after contact with the hypo-osmotic medium, the tails of some spermatozoa will begin to coil or swell. Tail swelling or curling indicates that the spermatozoon is undergoing hypo-osmotic stress, the plasma membrane is functional, and the cell is still viable. The spermatozoon is then picked up in the ICSI micropipette and placed in the other extra drop of medium in order to wash off excess hypo-osmotic medium from both the micropipette and the spermatozoon. The spermatozoon is then placed in the PVP drop in order to proceed with ICSI.
The choice and application of the appropriate sperm preparation technique can be a major contributor in influencing whether a patient will become pregnant. Ejaculates from subfertile males very frequently contain or have the potential for producing ROS, which are known to compromise sperm function and damage DNA. Therefore, it is imperative that the laboratory technologist selects a technique that will directly separate functional and highly motile spermatozoa from all that remains. Although it might be argued that density gradient centrifugation is the most effective method, not all laboratories may have access to the equipment and/or resources needed to perform the technique. Thus, depending on the initial sperm parameters, the direct swim-up from semen can safely and effectively be used and the post-swim-up centrifugation step may be omitted.
Although every specimen is considered valuable, oftentimes the specimens being handled are precious and/or expected to serve as a resource for many attempts at paternity. Thus, one must be even more selective when choosing a sperm-preparation technique. For example, a male undergoing medical therapy may be producing his last sperm-containing ejaculate, so optimization in the total number of motile sperm recovered is necessary. Further, due care is advised when processing an operatively obtained specimen, as repeat surgery exposes the patient to additional (and unnecessary) risk.
After reading this piece, one may ask: What kind of techniques might be on the horizon? Current technologies have afforded new knowledge about sperm biology. Expression of membrane proteins is not only a reflection of properly functioning spermatogenesis and spermiogenesis, but also sperm maturation. This characteristic may be exploited by using techniques that isolate cells based on the ionic charge of expressed membrane proteins (i.e., electrophoretically) or by using reversible binding techniques that involve cell function.
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