Sperm separation techniques
Under in vivo conditions, potentially fertile spermatozoa are separated from immotile spermatozoa, debris and seminal plasma in the female genital tract by active migration through the cervical mucus 5. During this process, not only progressively motile spermatozoa are selected, but male germ cells also undergo physiological changes called capacitation, which are fundamental prerequisites for the sperm's functional competence with regard to acrosome reaction 67.
The introduction of assisted reproduction, especially of IVF, during the 1980's, led to the development of a wide range of different sperm separation methods. Following the development of the classical swim-up method by Mahadevan & Baker 4, more complicated techniques were developed to increase the number of motile spermatozoa even in severe andrological cases. On principle, these techniques can be differentiated in migration, density gradient centrifugation and filtration techniques. For all migration methods, the self-propelled movement of spermatozoa is an essential prerequisite, while for density gradient centrifugation and filtration techniques the methodology is based on a combination of the sperm cells' motility and their retention at phase borders and adherence to filtration matrices, respectively. The migration techniques can again be subdivided into swim-up, under-lay and migration-sedimentation methods 89. For density gradient centrifugation, separation media like Ficoll® 10, Nycodenz 11 and Percoll® 1213 including the products (IxaPrep®, PureSperm®, Isolate®, SilSelect®) have recently been introduced to replace Percoll® 1415. The filtration methods like glass wool filtration 1617 and filtration of spermatozoa on Sephadex beads 18 and membranes 19 are alternative techniques.
The ideal sperm separation technique should (i) be quick, easy and cost-effective, (ii) isolate as much motile spermatozoa as possible, (iii) not cause sperm damage or non-physiological alterations of the separated sperm cells, (iv) eliminate dead spermatozoa and other cells, including leukocytes and bacteria, (v) eliminate toxic or bioactive substances like decapacitation factors or reactive oxygen species (ROS), and (vi) allow processing of larger volumes of ejaculates. Since none of the methods available meets all these requirements, a variety of sperm separation techniques is mandatory in clinical practice to obtain an optimal yield of functionally competent spermatozoa for insemination purposes. Depending on the ejaculate quality, these methods have different efficiency and areas of use. In the conventional swim-up technique, functional spermatozoa can come into close cell-to-cell contact with defective sperm or leukocytes by centrifugation, thus causing massive oxidative damages of the sperm plasma membrane by ROS and consequently of sperm functions 20. Therefore, the quality of the ejaculates has direct consequences on the choice of a sperm separation method.
With regard to the possible risk of a seroconversion in women and in the offspring after performing ART with spermatozoa from a HIV-positive man initial concerns arose as HIV viruses reportedly can bind to and penetrate into spermatozoa 2122. Later research, however, showed that HIV-1 genomes found to an extend of 18% in seminal cells samples decreased to undetectable levels following a combined density gradient centrifugation with a subsequent swim-up 23. No seroconversion of the virus could be observed after fertilization of oocytes by IVF-ICSI 242526. Other recent research 27 showed that HIV RNA and DNA could be detected in separated spermatozoa even in treated patients. Thus, viral validation of separated spermatozoa is necessary and should be performed even in treated patients. Additionally, only HIV-tested maternal serum or commercially available serum albumin, which is HIV-free due to its processing, should be used as protein supplement for culture media.
In order to give an overview, only the four most common techniques classical swim-up, migration-sedimentation, density gradient centrifugation and glass wool filtration are discussed.
Swim-up procedure
Apart from a simple wash and subsequent resuspension of the male germ cells, the swim-up from a washed pellet is the oldest and most commonly used sperm separation method. Originally described by Mahadevan and Baker 4, this method is still used largely in IVF laboratories around the world. Although its use among the male factor infertility group is very limited, the swim-up is still the standard technique for patients with normozoospermia and female infertility. Excellent fertilization rates were reported when these sperm preparations were used to inseminate human oocytes in vitro. However, as the indications for IVF were expanded beyond simple tubal factor cases to couples with idiopathic infertility and, ultimately, to male factor cases, the problem of fertilization failure appeared 282930.
The methodology of this conventional swim-up is based on the active movement of spermatozoa from the pre-washed cell pellet into an overlaying medium. Typically, the incubation time is 60 minutes. This technique is distinguished by a very high percentage (>90%) of motile sperm, preferred enrichment of morphologically normal spermatozoa as well as the absence of other cells and debris. Considering that the efficiency of the technique is based on the surface of the cell pellet and the initial sperm motility in the ejaculate, the yield of motile spermatozoa is limited. Many layers of cells in the pellet may cause potentially motile spermatozoa in the lower levels of the pellet never to reach the interface with the culture medium layer. In addition, a significant decrease in the percentage of normally chromatin-condensed spermatozoa has been reported after the swim-up procedure 31. Another major disadvantage of this technique is the fact that for its use spermatozoa are pelleted, thus coming into close cell-to-cell contact with each other, cell debris and leukocytes, which are known to produce very high levels of reactive oxygen species (ROS) 32. Due to the extraordinary high amount of poly-unsaturated fatty acids in the sperm's plasma membranes 33, these ROS cause lipid peroxidation and therefore a dramatic decrease in sperm functions, including motility 34. Overall, although many men's spermatozoa may not be impaired to the extent of inhibiting fertilization, some couples' chances of successful IVF will certainly be compromised. It is therefore not reasonable to continue and to use a technique, such as swim-up from pelleted semen with the inherent potential to cause irrevocable damage to spermatozoa prejudicial to a desired functional endpoint. Eventually, this knowledge led to the development of other more gentle sperm separation methods that also allow a higher recovery of motile and functional spermatozoa. The advantages and disadvantages of the conventional swim-up are summarized in table 1.
<p>Table 1</p>
Advantages and disadvantages of the conventional swim-up method.
Advantages
Disadvantages
- easy to perform
- restricted to ejaculates with high sperm count and motility
- very cost-effective
- usually recovery of a very clean fraction of highly motile spermatozoa
- low yield
- spermatozoa can be massively damaged by reactive oxygen species
- significant decrease of the percentage of normally chromatin-condensed spermatozoa
An attempt to overcome at least the problems caused by ROS, the "swim-up" can be performed directly from the liquefied semen. During this procedure, several aliquots of liquefied semen are taken from a sample and placed in tubes underneath an overlay of culture medium. Round-bottom tubes or 4-well dishes should be used to optimize the surface area of the interface between the semen layer and the culture medium. The tubes may also be prepared by gently layering culture medium over the liquefied semen. The placing of semen underneath the culture medium, however, provides a much cleaner interface zone. A maximum recovery is obtained by using multiple tubes with small volumes of semen per tube, thus maximizing the combined total interface area between semen and culture medium. Mortimer 35 suggested the use of 250 μl semen and 500 to 600 μl culture medium per tube. After the incubation period, which is typically between 30 and 60 minutes, at 37°C, most of the upper culture medium layer is removed. This should be done with caution, working from the upper meniscus downwards, using a sterile pipette. Typically, 75 or 80% of the culture medium layer are removed and eventually combined, taking great care not to aspirate directly from the interface region. This procedure will also increase the total number of recovered spermatozoa, which can then also be used for ICSI 36.
Other swim-up methods include the swim-up of spermatozoa in a specially supplemented medium. Such substances can be SpermSelect™, which is a highly purified preparation of hyaluronic acid (Pharmacia, Uppsala, Sweden) with an average molecular weight of 3,000 kDa that was used at a final concentration of 1 mg/ml in culture medium. Compared with the traditional swim-up from a washed pellet 37, a swim-up directly from semen into a hyaluronic acid solution gave a significantly higher percentage of motile spermatozoa and, ultimately, the achievement of a higher pregnancy rate in a clinical IVF program 38. However, highly purified hyaluronic acid is expensive and it has been shown to increase the calcium-influx into spermatozoa and therefore induce acrosome reaction 39. Elevated local concentrations of hyaluronic acid in the cumulus oophorus have been shown to contribute to the acrosome reaction 40. Thus, it seems rather questionable whether this substance is favourable for IVF. In addition, whether these improved results in sperm motility were specifically due to the use of the hyaluronate or to the use of a method, which did not involve the initial pelletting of unselected spermatozoa, has not been ascertained. On the other hand, however, hyaluronic acid has been regarded as an effective alternative to test sperm penetration into human cervical mucus 414243.
Migration-sedimentation
A more sophisticated sperm separation technique is migration-sedimentation, which was developed by Tea et al. 8. Principally, this method is a swim-up technique combined with a sedimentation step. Special glass or plastic tubes with an inner cone are used. In contrast to the conventional swim-up procedure, spermatozoa swim up directly from liquefied semen into the supernatant medium and subsequently sediment in that inner cone within an hour's time. Thus, this method is a highly gentle technique, especially if compared with methods that require centrifugation steps before the sperm separation like the conventional swim-up. In the original version, a fraction of highly motile and functionally competent spermatozoa can be obtained. Unfortunately, the yield is very low and therefore the original method did not find wide acceptance for IVF. The advantages and disadvantages of this method are summarized in table 2. Recently, Zavos et al. 9 proposed the use of a multi-chamber tube to retrieve functional spermatozoa for assisted reproductive techniques by means of a swim-up and sedimentation method. The assessment of its usefulness for IVF/ICSI, however, is still to be awaited.
<p>Table 2</p>
Advantages and disadvantages of the original migration-sedimentation method according to Tea et al. (1984).
Advantage
Disadvantages
- usually very clean fraction of highly motile spermatozoa
- the original method is restricted to ejaculates of high sperm count and good motility
- reactive oxygen species are reduced
- the original method has a very low recovery rate
- very gentle separation method
- special glass or plastic tubes are required
- tubes are more expensive and relatively sensitive
- for repeated use in IVF, glass and plastic tubes must be sterilized
Sánchez et al. 44 showed that after concentration of sperm cells in the ejaculate, even in cases with severe oligo- and/or asthenozoospermia a sufficient number of motile spermatozoa can be isolated for intracytoplasmic sperm injection (ICSI) after 2–3 hours of incubation. Compared with the density gradient centrifugation, these authors also demonstrated significantly better results for progressive motility, normal sperm morphology, chromatin condensation and reduction of the percentage of dead spermatozoa as determined by the eosin test. In addition, since spermatozoa isolated by this modified migration-sedimentation technique stick markedly less to the glass surface of the injection pipettes, this method has even an advantage over the density gradient centrifugation, which is normally used for these cases. In this regard, the side migration technique that was recently proposed by Hinting and Lunardhi 45 is another interesting approach to obtain motile spermatozoa from very poor quality semen for ICSI as it also yielded better sperm quality.
Density gradient centrifugation
The typical methodology for the density gradient centrifugation comprised continuous 46 or discontinuous gradients 47. With continuous gradients, there is a gradual increase in density from the top of the gradient to its bottom, whereas the layers of a discontinuous gradient show clear boundaries between each other. The ejaculate is placed on top of the density media with higher density and is then centrifuged for 15–30 minutes. During this procedure, all cells reach the semen sediment. However, highly motile spermatozoa move actively in the direction of the sedimentation gradient and can therefore penetrate the boundary quicker than poorly motile or immotile cells, thus, highly motile sperm cells are enriched in the soft pellet at the bottom.
A wide variety of methods using the principle of density gradient centrifugation to fractionate subpopulations of spermatozoa has been described in the literature. Ficoll® has initially been used as gradient material for preparing spermatozoa 48, but by far the most widely used substance for all methods of assisted reproduction (IUI, GIFT, IVF, ICSI, etc.) have been the polyvinylpyrrolidone (PVP)-coated silica particles Percoll®. Normal sperm function in terms of sperm fertilizing ability as assessed in the zona pellucida-free hamster egg penetration test 2049, as well as in human IVF 13 and ICSI 50 was observed. In October 1996, Percoll® has been withdrawn from the market for clinical use in assisted reproduction 51. This was because of the risk of contaminations with endotoxins 525354, possible membrane alteration 5556 and inflammatory responses that could be induced by the insemination of sperm populations contaminated with Percoll®. In addition, Percoll® adheres to the sperm membranes 57 and might alter them by removing coating envelopes 58. Therefore, intensive washing of the spermatozoa after sperm separation with Percoll® was recommended 55. This requires additional centrifugation and can again be detrimental to the spermatozoa because of the action of reactive oxygen species 20.
Another commercial product known as Nycodenz (Nyegaard & Co., Oslo, Norway) was also used as a density gradient material for preparing human spermatozoa. Nycodenz is the same molecule, iohexol, as used in the X-ray contrast medium Omni-paque. Studies revealed a low incidence of adverse reactions during angiography 11. Both continuous and discontinuous Nycodenz gradients were evaluated, of which a four-layer discontinuous gradient was found to produce populations of highly motile spermatozoa with better yields and survival than either swim-up or Percoll® gradients from oligozoospermic and asthenozoospermic semen samples 1159. Compared with the conventional swim-up procedure from a pelleted sperm population, the use of Nycodenz also seems to be superior regarding sperm penetration into zona-free hamster eggs 60. Thus, this technique has clearly great potential in the preparation of motile spermatozoa from poor quality semen for IVF use and warrants further investigation.
Other replacement products for Percoll® that were introduced into the market from the mid nineties and more commonly used in assisted reproduction are IxaPrep® (MediCult, Copenhagen, Denmark), SilSelect® (FertiPro N.V., Beernem, Belgium), PureSperm® (NidaCon Laboratories AB, Gothenburg, Sweden) or ISolate® (Irvine Scientific, Santa Ana, CA, USA). In contrast to Percoll®, which is a PVP-coated silica that can have deleterious effects on sperm membranes 56, all these replacement products contain silane-coated silica particles, are adjusted for the osmolarity with polysucrose and have very low toxicity. All these replacement products are non-irritating and are approved for human in vivo use. The results of sperm preparation using these new products compared with Percoll® regarding recovery rate, motility, viability, normal sperm morphology and velocity parameters like VAP or VCL vary considerably among different working groups. While Claassens et al. 61 and Söderlund and Lundin 62 did not find differences in the recovery rate between Percoll® and PureSperm®, Chen and Bongso 63, depending on the number of layers included for the density gradient, reported significantly higher values for PureSperm®. For IxaPrep® it is even more confusing because Yang et al. 64 found no difference to Percoll®, while Makkar et al. 14 found the replacement substance more effective. On the other hand, McCann and Chantler 65 as well as Ding et al. 66 found Percoll® superior. These authors attribute the better sperm quality obtained after the IxaPrep® preparation to a significantly decreased production of nitric oxide, which is regarded as sperm toxicant that reduces motility 67. This could be due to an activation of guanylyl cyclase, thus increasing cGMP production, which inhibits sperm motility 68. On the other hand, nitric oxide is also known to be a physiologic mediator for vasodilatation, immunosuppression, neurotransmission and cytotoxicity 69707172.
Regarding the other parameters such as motility, viability, normal sperm morphology or velocity parameters like VAP, the data currently available also vary considerably among different working groups. The reason for this can be attributed to the different conditions of the sperm separation, e.g. volume of semen to be separated, g-force, centrifugation time or the number of layers of the gradient, and reflect the important role of the methodology. Moreover, this also shows that these data cannot be directly compared. Overall, the Percoll®-replacement products are good and reasonable alternatives, and this not only for the fact that Percoll® is no longer allowed to be used for clinical purposes in assisted reproduction.
Glass wool filtration
During glass wool filtration, which has already been described by Paulson & Polakoski in 1977 17, motile spermatozoa are separated from immotile sperm cells by means of densely packed glass wool fibres. The principle of this sperm separation technique lies in both the self-propelled movement of the spermatozoa and the filtration effect of the glass wool. The success of this method is directly linked to the kind of glass wool used 73. Thus, factors like the chemical nature of the glass (i.e. borate glass, silicate glass or quartz glass), the surface structure and charge of the glass wool, thickness of the glass wool fibres or the pore size of the filter have to be taken into consideration. In clinical practice, the glass wool, code number 112, from Manville Fiber Glass Corp. (Denver, CO, USA) or SpermFertil® columns from Mello (Holzhausen, Germany) have been tested extensively. Potential risks of the technique such as damages of the spermatozoa or the occurrence of glass wool fragments in the filtrate essentially depend on the kind of glass wool used and on the intensity of the washing prior to the filtration.
Compared with the swim-up or migration-sedimentation, glass wool filtration, just as density gradient centrifugation, is a technique that uses the whole volume of the ejaculate and therefore yields a significantly higher total number of motile spermatozoa 3174. Thus, it can also be used for patients with oligo- and/or asthenozoospermia 74. Like density gradient centrifugation, glass wool filtration also provides the advantage that the sperm separation can directly be performed from the ejaculate. Only after the separation of the functional spermatozoa from the immotile ones, leukocytes and debris, a centrifugation step will be necessary to remove the seminal plasma. This is an important aspect as this procedure reduces cellular damage by reactive oxygen species. The advantages and disadvantages of this method are summarized in table 4.
<p>Table 4</p>
Advantages and disadvantages of the glass wool filtration.
Advantages
Disadvantages
- simple to perform
- a bit more expensive
- normally, recovery of spermatozoa with good motility
- the filtrate is not as clean as it is with other sperm separation methods
- spermatozoa from ejaculates with a very low sperm density can be separated
- remnants of debris are still present
- good yield
- leukocytes are eliminated to a large extent
- reactive oxygen species are significantly reduced
By means of glass wool filtration, it is even possible to prepare motile spermatozoa from patients with retrograde ejaculation (Henkel et al., unpublished). In these cases, the procedure includes adjustment of the osmolarity of the patient's urine to values of about 350 mOsmol/kg by drinking water. Prior to the ejaculation, the patients are requested to urinate most of the urine in the bladder. The small amount of antegrade-produced ejaculate is collected in a plastic beaker, while the retrograde fraction of the ejaculate needs to be urinated immediately into a jar with 50 ml culture medium containing human serum albumin to dilute the urine, Finally, the urine/medium mixture has to be centrifuged, resuspended in 3 to 4 ml of fresh medium and filtrated on the glass wool column. As constituents of the urine can damage the spermatozoa, a speedy work-up of such ejaculates is mandatory.
In addition to the separation of spermatozoa, glass wool filtration has been shown to eliminate leukocytes to an extent of up to 90% 75. Since leukocytes are frequent even in normal ejaculates 76 and produce 100-times more ROS than spermatozoa 32, this effect significantly contributes to a reduction of free radicals in the ejaculate 7375. This is of paramount importance for the functionality of spermatozoa because the male germ cells are particularly susceptible to oxidation by ROS because of their extraordinary high content of polyunsaturated fatty acids in their plasma membrane 337778.
Another clinically interesting aspect related to glass wool filtration is chromatin condensation, which has repeatedly been shown to be predictive of fertilization in vitro 798081. Glass wool filtration 31 like the density gradient centrifugation with PureSperm® 82 or the migration-sedimentation technique 44 significantly selects normally chromatin-condensed spermatozoa, while conventional swim-up or Percoll®-centrifugation decrease this sperm parameter. As human sperm chromatin condensation follows a seasonal rhythm, which even shows a shift of about half a year on the southern hemisphere 83, this might have a clinical impact on the results in IVF. Should a patient be examined in winter when the quality of sperm chromatin condensation is high 83 and referred to IVF in summer when the percentage of normally chromatin-condensed spermatozoa is significantly lower, IVF for this patient might fail. Thus, for these patients a sperm separation by means of glass wool filtration, PureSperm® or migration-sedimentation might be beneficial.
Glass beads
This method has been used for the preparation of hamster spermatozoa for in vitro capacitation 84 and resulted in an efficient, high yield selection of motile human spermatozoa from semen 85. However, there were concerns regarding the possible spill over of beads into the insemination medium. As a result, the use of glass beads for effective sperm preparation for assisted reproduction has not widely been accepted.
Sephadex columns
In the early nineties sperm separation by means of Sephadex beads emerged 18 and a commercial sperm separation kit based on this principle (SpermPrep®) has become available. Compared to migration-sedimentation and swim-up from pelleted semen it produced significantly higher yields 86. Moreover, morphologically normal sperm cells could be enriched in the filtrate after SpermPrep® separation as well as significantly higher pregnancy rates for intrauterine insemination as compared with the conventional swim-up method 87. In a comparison between SpermPrep® method and Percoll® centrifugation, Percoll® separated spermatozoa showed a significantly higher percentage of normally chromatin-condensed and morphologically normal spermatozoa 88. However, the fertilization rates reported by these authors were similar. López et al. 89 used a prepacked PD-10 column containing Sephadex G-25 particles (Pharmacia Biotechnology, Uppsala, Sweden), which is normally used to desalt proteins in solutions, to separate human spermatozoa and compared the results with the SpermPrep® method and Percoll® centrifugation. The PD-10 column and density gradient centrifugation in Percoll® yielded a comparable number of spermatozoa and showed similar percentages of morphologically normal spermatozoa after sperm separation. On the contrary, the SpermPrep® method resulted in significantly lower values of sperm count and morphology.
Transmembrane migration
Another alternative sperm separation technique that was also developed in the late eighties is migration/filtration of motile spermatozoa through a Nuclepore membrane filter. These filters are unusual because their pores are cylindrical and at right angles to the plane of the membrane 90. The spermatozoa, therefore, have straight channels to swim through the membrane. Unfortunately, these membranes had a very low ratio of the total cross-sectional area of the pores to the overall membrane area. Consequently, the yield is extremely low. Primarily, this method was used for testing the motility of sperm populations treated with various pharmacological agents, but not as a preparation method for assisted reproduction 91.
Another approach of separating viable human spermatozoa by means of membranes was undertaken by Agarwal et al. 19 using a membrane which has been developed for selective removal of leucocytes (L4 membrane). Besides a significant increase of motility, ejaculates filtered through this membrane have been shown to contain fewer leukocytes. This fact is, of course, of importance in those cases that have increased numbers of leucocytes in the ejaculate as a result of infections. Moreover, this membrane seems to be selective for spermatozoa with normal membrane integrity 9293 and sperm producing low amounts of reactive oxygen species 94. However, despite these advantages of the membrane it has never come into practical clinical use for human assisted reproduction.
In vitro treatment of spermatozoa
An alternative and/or complementary approach to sperm preparation for ART in vitro by means of the above discussed sperm separation methods is the idea to treat the spermatozoa in vitro in order to improve their functionality, i.e. motility, or to supply a protective environment with the purpose to maintain or improve their functional capacity for successful fertilization. Many substances including serum, peritoneal fluid and follicular fluid or other chemically defined pharmacological substances like progesterone, adenosine analogues or methylxanthins have been proposed to stimulate human sperm functions. The methodologies addressing the different aspects, motility and sperm functions, ROS, and the reduction of the visco-elastic properties of the semen are discussed below.
Improvement of motility and sperm functions
CAFFEINE
The use of xanthine (Fig. 1A) derivatives like the methylxanthines caffeine (1,3,7-trimethyl-2,6-dioxopurine) (Fig. 1B) and pentoxifylline (Fig. 1C) for the pharmacological stimulation of sperm functions, especially for motility, is well known. Garbers et al. 109 already suggested stimulation of human sperm motility by caffeine. This substance is an inhibitor of the phosphodiesterase that leads to increased cellular levels of cAMP. Considering that a defective energy metabolism of the spermatozoa is also a potential cause of male infertility, the use of motility stimulating substances to improve sperm motility and therefore fertilization rates appeared reasonable.
<p>Figure 1</p>
Molecular structures of xanthine (A), caffeine (B), pentoxifylline (C) and 2-deoxyadenosine (D)
Molecular structures of xanthine (A), caffeine (B), pentoxifylline (C) and 2-deoxyadenosine (D).
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Human spermatozoa can obtain energy from both glycolysis and mitochondrial oxidative phosphorylation 110. Thus, motility parameters such as mode of progression, lateral head displacement or velocity of fresh and cryopreserved spermatozoa significantly increased after stimulation with caffeine due to augmented glycolysis and fructolysis 111112113. A small study including 6 patients by Serres et al. 114 revealed that the stimulatory effect is most obvious in patients with asthenozoospermia. However, early reports documented not only a stimulating effect of caffeine, but also a time- and concentration-dependent detrimental effect on the sperm plasma membrane 115116. Moreover, spermatozoa pre-incubated with caffeine showed a significant decrease in the percentage of penetrated zona-free hamster oocytes 117. The only study available 118 that investigated the effect of caffeine in assisted reproduction using 42 oocytes from 21 women showed an increase in sperm motility but a significant decrease in the fertilization rates and embryo development.
Direct incubation of mouse oocytes and embryos resulted in a time- and concentration-dependent artificial parthenogenic activation of the oocytes and a significant reduction of embryo cleavage rates 119. As these data were more disappointing than stimulating for the clinical use of caffeine in assisted reproduction, it was further only used for research purposes. A very recent prospective study by Klonoff-Cohen et al. 120 investigated the daily caffeine consumption of 221 patients and its effects on the outcome of IVF/GIFT treatments. According to these authors, failed pregnancies were significantly associated with the female's caffeine consumption. In addition, gestational age at delivery decreased significantly in women who consumed more than 50 mg/day. Male caffeine consumption appeared to be a significant risk factor for multiple gestations but had no effect on sperm count, motility or normal sperm morphology. An earlier study 121, however, demonstrated a positive association between coffee consumption and sperm concentration, motility and abnormal sperm morphology in a total of 546 men. In combination with smoking as further risk factor, sperm motility and viability decreased significantly. Klonoff-Cohen et al. 120 confirmed the embryotoxic effect of caffeine causing delayed conception 122 or spontaneous abortions 123 and concluded that the caffeine consumption should be minimized prior to and during an assisted reproduction treatment.
PENTOXIFYLLINE
Pentoxifylline (Fig. 1C) is another methylxanthine derivative, which is also, like caffeine, a non-specific inhibitor of phosphodiesterase. In contrast to caffeine, the Food and Drug Administration (FDA) approved pentoxifylline for the administration to humans. The drug is used for systemic treatments of patients with cardio-vascular diseases (Trental® or Torental®). In addition, since its water solubility is higher than that of caffeine, this increases its usability 124.
The beneficial effect of pentoxifylline on sperm motility and motion characteristics like sperm velocity or hyperactivity has repeatedly been described for both fresh 112125126127128129 and cryopreserved spermatozoa 128130131132. The results on the stimulation of sperm motility are conflicting. Yovich et al. 133, Rees et al. 112 and Lewis et al. 134 found no effect in normozoospermic patients, while others 133134135136 observed a significant increase in motility and the number of progressively motile spermatozoa in patients with asthenozoospermia. This stimulatory effect can clearly be attributed to the increased intracellular levels of cAMP. In the absence of the drug, the cAMP content remained unchanged and correlated only with hypermotility and the amplitude of the lateral head displacement of the spermatozoa 137. Cyclic AMP, in turn, is believed to stimulate a cAMP-dependent kinase 138, which itself induces sperm tail protein phosphorylation 139 with subsequent increase in sperm motility 140.
Apart from the effects on sperm motility, pentoxifylline is also reported to augment acrosome reaction 141. Nassar et al. 142 demonstrated that this induction is not due to a Ca2+-influx in the sperm cell, which is regarded to stimulate acrosome reaction 143. The intracellular [Ca2+]i even decreased following the pentoxifylline treatment. Since cAMP is intimately involved as a second-messenger in the induction of acrosome reaction 144, an unspecific inhibition of phosphodiesterases by this methylxanthine will increase the intracellular cAMP levels and therefore induce acrosome reaction. In addition, this drug also improves the spermatozoa's zona pellucida binding ability 139145146. However, it appeared that this binding improvement to the zona pellucida is rather a result of the increase in the sperm velocity parameters straight-line velocity (VSL) and average path velocity (VAP) as these parameters are indicators of progressive motility. Therefore, these spermatozoa represent a sperm population that did not initiate acrosome reaction with its characteristic change in movement characteristics yet 147.
The benefits of a treatment with pentoxifylline prompted its use in assisted reproduction programs. However, the results reported in the literature are rather conflicting. While Tasdemir et al. 148 and Tarlatzis et al. 149 found an improvement in the IVF rate and saw a promising development in small studies comprising 51 and 43 patients, respectively. Others did not find differences between the control and the treatment group in IVF and intrauterine insemination 150151152. Thus, it was concluded that pentoxifylline should not be used indiscriminately 153. On the other hand, pentoxifylline has been successfully used to increase fertilization rates in bovine in vitro fertilization 154 and as pre-treatment to stimulate epididymal and testicular sperm motility for ICSI 155156. Should the results of Numabe et al. 154 be confirmed, this could be a promising approach to improve fertilization rates especially for endangered species.
The conflicting results on the effectiveness of a pentoxifylline treatment raised the question of the embryotoxicity of this substance, especially since possible pentoxifylline-induced adverse effects on spermatozoa 157 and mouse embryo development 158159 have been reported. In contrast, Lacham-Kaplan & Trounson 160 did not observe such negative effects on embryonic development after insemination of the oocytes with spermatozoa incubated in 3 mM pentoxifylline. Finally, short-term incubation of spermatozoa with subsequent washing of the male germ cells did not produce such adverse effects in intrauterine insemination or ICSI (Henkel et al., unpublished) 156. An alternative approach to increase sperm motility or the number of motile spermatozoa was to administer the drug orally over period of 3 to 6 months 161. In a placebo-controlled study including 47 normozoospermic men with idiopathic asthenozoospermia, Merino et al. 162 showed a significant increase in progressive motility in men who received 1,200 mg of pentoxifylline per day over 6 months. Clinical data about fertilization and pregnancy, however, are still not available.
Another important point that must not be underestimated in explaining the controversial effects of pentoxifylline is the fact that this drug is an unspecific inhibitor of the phosphodiesterase (PDE). Considering that eleven different families of this enzyme have been described 163, of which PDE-1 and PDE-4 are present in human spermatozoa and stimulate different sperm functions, i.e. acrosome reaction and motility, respectively 164, an unspecific inhibition of the PDE's will obviously result in both, stimulation of motility and acrosome reaction. Depending on the conditions and most importantly on the time of stimulation and the concentration of pentoxifylline in the medium, over-stimulation will definitely result in a too early acrosome reaction. Hence, over-stimulated spermatozoa for an IUI or IVF treatment will not fertilize oocytes because they are no longer able to bind to the zona pellucida. This dilemma might be overcome by the use of a non-embryotoxic PDE-4 inhibitor to stimulate sperm motility only. Unfortunately, to our knowledge, no further progress has been made in this regard. For ICSI, this problem is not relevant as spermatozoa bypass all physiological barriers because they are directly injected into the oocytes.
2-DEOXYADENOSINE
2-Deoxyadenosine (2-DA) (Fig. 1D) is an adenosine derivative that is not a phosphodiesterase inhibitor like pentoxifylline or caffeine. Like these substances, 2-DA is also a potent stimulant of sperm motility 165166. The molecular mechanism of motility stimulation, however, works via an A2-receptor-mediated activation of adenylate cyclase 167, which in turn is thought to enhance the intracellular cAMP concentration 168169. Because of this characteristic, the use of 2-DA in assisted reproduction programs has been discussed. Other studies, however, failed to detect a responsive cAMP activity and either stimulatory or inhibitory G-proteins in spermatozoa 170171. In addition, Rivkees 172 could not detect A2a- and A2b-receptor gene expression in the rat testis. This would imply that these stimulatory receptors are either not present or, at least, at very low levels. On the other hand, the cAMP-inhibitory A1-receptor has a capacitative effect on human spermatozoa when stimulated with an agonist 173. While Imoedemhe et al. 174 in an internally controlled prospective study showed significantly higher fertilization rates after sperm stimulation with 2-DA and hence suggested further evaluation of the drug in assisted reproduction programs, Tournaye et al. 150 recommends a careful evaluation and selection of the patients before the treatment with motility stimulants.
Recently, 2-DA and pentoxifylline have been suggested for sperm stimulation in an in vitro culture of testicular tissue in order to obtain motile spermatozoa for ICSI 175. However, it seems rather questionable whether a treatment of spermatozoa with 2-DA for the purpose to fertilize oocytes should be recommended because there is also evidence that it has adverse effects on embryos. In this regard, 2-DA significantly reduced the cleavage of mouse embryos beyond the 2-cell stage 160. In addition, 2-DA and cAMP have been shown to exert cytotoxic effects by inducing G1 cell cycle arrest 176. In view of this, a therapeutic clinical use of stimulants that increase the intracellular cAMP levels should be evaluated very carefully. At least, a very careful washing of the spermatozoa is mandatory.
KALLIKREIN
A substance, which has been discussed very controversially regarding its stimulatory effect on sperm motility and sperm functions during the past ten years, is kallikrein. Despite all components of the kallikrein-kinin system are present in the male and female genital tract 177178 and the localization of the bradykinin B2-receptor in rat testis 179180, the function of the kallikrein-kinin system for male reproductive function is still unclear. The prostate-specific human glandular kallikrein, which is about 500-times less effective than tissue kallikrein, is present in human seminal plasma 181. In addition, in vitro studies reported a positive effect of kallikrein and its cleavage products, the kinins, on sperm functions including motility 182183. This suggests an involvement of this enzyme or bradykinin in the male reproductive system. Gerhard et al. 184 reported a significant improvement of sperm motility when using kallikrein in an artificial insemination program where 172 patients were randomly assigned to the treatment and control group. However, the penetration distance of sperm in cervical mucus was significantly lower and was regarded as cause for the lower pregnancy rates in this group of patients. In contrast, Schill et al. 185 found an increased cervical mucus penetrability of human spermatozoa following treatment of with hog pancreatic kallikrein and bradykinin.
The results regarding ART are as contradictory as the results on in vitro stimulation of spermatozoa. Schill and Littich 186 reported an increased pregnancy rate in intrauterine insemination following stimulation of spermatozoa with kallikrein in a cross-over blind study in 48 asthenozoospermic and oligoasthenozoospermic therapy-resistant patients in 468 inseminations. All patients included in that study were shown to respond to this treatment beforehand. On the other hand, there are in vitro studies 187188 and double-blind placebo-controlled studies 189190 that did not show any effect of kallikrein and bradykinin on sperm motility or sperm count. Miska et al. 191 showed that porcine pancreatic kallikrein, which is taken orally, is absorbed in unaltered form by the intestine. In the light of the recent data by Monsees et al. 178179180 obtained in the rat, the kallikrein-kinin system in the male reproductive tract seems to play a role in the regulation of Sertoli cell function, the local regulation of spermatogenesis or in the function of the seminiferous tubules.
BICARBONATE
Another interesting approach to improve sperm motility and thus sperm recovery from the ejaculate that has not yet been used for clinical application in assisted reproduction is the stimulation of the sperm cell's kinematics by bicarbonate 192. It is well known that bicarbonate is a major secretory component of the fallopian tube that stimulates sperm respiration 193, and is also postulated to be beneficial for fertilization 194. The latter appears to be supported by its effect on capacitation, induction of acrosome reaction 192195196 and hyperactivated motility, which in turn is required for successful zona penetration in the hamster 197. These physiological changes, especially acrosome reaction and hyperactivation, require the influx of Ca2+ into the spermatozoa. Acrosome reaction is absolutely dependent on the presence of extracellular calcium 195. Recent work by Wennemuth et al. 198 showed that bicarbonate also facilitates the opening of voltage-gated Ca2+-channels, which are eventually involved in the increase in flagellar beat frequency shortly after stimulation. Thus, bicarbonate is an important mediator of sperm cell function.
Therefore, media with enhanced levels of this anion might be helpful for sperm preparation and assisted reproduction. Henkel et al. 199 showed that sperm preparation by using a medium containing high levels of bicarbonate resulted in a significantly higher progressive motility as well as sperm recovery. In addition, the co-incubation of human spermatozoa with zonae in this medium resulted in a significantly increased zona binding of the spermatozoa. Jaiswal and Majumder 200 made similar observations in testicular and epididymal spermatozoa from goat and ram. While theophylline, a phosphodiesterase inhibitor, and epididymal fluid only induced a non-progressive flagellar movement of these initially immotile sperm, the addition of bicarbonate induced forward motility in 16 to 40% of the sperm cells. This motion stimulating effect of bicarbonate, which otherwise only appears in a subpopulation of the spermatozoa 201, is mediated by an activation of adenylate cyclase 202203 with subsequent increased levels of cAMP, which in turn stimulate protein kinase A 204 and results in protein phosphorylation 205. H89, a highly specific inhibitor of protein kinase A, significantly inhibited bicarbonate induced sperm motility and indicates the importance of this enzyme for sperm motility 201.
Considering motility and acrosome reaction as important sperm functions and bicarbonate as mediator as well as the aspect that this anion is non-toxic as other motility stimulants, it appears tempting to use a high bicarbonate medium not only for diagnostic purposes 199, but also in a clinical approach to improve sperm motility and functions for assisted reproduction. However, as elevated pH levels can disturb the mitotic spindle, a treatment of oocytes with bicarbonate should be performed with high care.
CHELATORS
In a very recent approach, Wroblewski et al. 106 investigated the influence of different metal chelators, i.e. DL-penicillamine, 2,3-dimercaptopropan-1-sulfonate and meso-2,3-dimercapto-succinimic acid, on human sperm motility in vitro. These authors showed that the percentage motility and velocity, even of swim-up separated spermatozoa, can be enhanced by incubation with these chelators, and speculate of a possible future use of such a procedure to improve fertilization rates in IUI or IVF.
The proposed mechanism of this motility enhancement is the removal of the element zinc from the outer dense fibres (ODF), which are functionally essential substructures in the mammalian sperm flagellum. During spermiogenesis, cysteine and zinc are incorporated in spermatozoa 207. In human spermatozoa, zinc is localized in the flagellum to an extent of 93–97% 208, especially in the ODF. When incorporated in the ODF, zinc is first associated with the sulfhydryl groups of cysteine by formation of relatively stable zinc-thiol complexes 209 in order to protect ODF from premature oxidation 210. This trace element is later removed from the sperm to an extent more than 60% during epididymal sperm maturation 207211. Thereafter, the sulfhydryl groups are oxidized to disulphide-bridges that stabilize and stiffen the ODF 212, which in turn leads to a better energy conversion and therefore to better motility including forward progression 213214.
Latest research from our working group, however, showed that at least DL-penicillamine and 2,3-dimercaptopropan-1-sulfonate are not suitable for clinical use as both chelators alter the sperm's responsiveness to the induction of acrosome reaction (Henkel et al., unpublished). Thus, it remains to be seen whether or not other chelators will not show such adverse effects.
PLATELET-ACTIVATING FACTOR (PAF)
Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine) is a biologically highly potent signalling ether phospholipid, which was first described by Benveniste et al. 215. Apart from its multiple functions on circulation, inflammation, systemic vasodilatation, pulmonary bronchiole constriction, platelet and neutrophile activation, cardiac ischemia, tissue rejection, gastric ulcer and development 216217218, it is reported to be a cellular mediator in reproduction. Here, PAF appears to be involved in implantation 219 and may reflect embryo health and viability 220221. Moreover, PAF has been found in spermatozoa of different species like rabbit, mouse, pig, rhesus monkey and human 221222223224225226.
Several authors reported positive effects on motility, capacitation, acrosome reaction and oocyte penetration 227228229230. PAF antagonists can inhibit its positive actions on sperm function 231. The sperm content of PAF in the species investigated is positively related with the fertility status of the male 232. Reinhardt et al. 233 provided evidence for a PAF receptor on human spermatozoa at the midpiece and proximal sperm head suggesting that the action of PAF is receptor-mediated, which does not result in increased levels of intracellular cAMP concentrations 234235. However, PAF binds to the surface receptor and activates a phospholipase, which in turn converts diacylglycerol (DAG) to inositol triphosphate (IP3), and thus increases the intracellular [Ca2+]i concentration that can either be released from intracellular stores or by an extracellular Ca2+-influx via Ca2+-channels. This intracellular Ca2+-increase may thus be responsible for the induction of acrosome reaction 236. However, although the exact molecular mechanism of the action of PAF has not yet been elucidated in detail, these positive effects led the use of PAF in assisted reproduction. Recently, in a prospective, randomized, blinded study in 143 patients, Roudebush et al. 237 presented data showing that pregnancy rates in intrauterine insemination were significantly improved after separating the spermatozoa with a medium containing PAF. The advantage of such a treatment is that PAF is a natural substance that is non-toxic as it is with many other motility stimulators.
Selection of live spermatozoa from a completely immotile sperm population prior to ICSI
An important aspect of sperm selection and the improvement of sperm functions are reflected by the selection of spermatozoa for ICSI from extremely poor semen quality. In this regard, special emphasis should be laid on the selection of spermatozoa from a completely immotile sperm population. For the success in ICSI it is important to inject viable spermatozoa. Normally, this distinction is made by the sperm's most obvious function, motility; a motile spermatozoon is live. However, in this particular group of patients this distinguishing mark is void and scientists were urged to find non-harmful methods that identify live spermatozoa in a population of immotile sperm and that are suitable for the use in ICSI, i.e. that do not harm the oocyte. To date, two different approaches for the distinction between live and dead spermatozoa have been pursued; (i) the initiation of motility as sign of vitality by means of stimulants and (ii) the identification of live spermatozoa according to their membrane integrity by means of the hypo-osmotic swelling test (HOS test).
The initiation of motility in an immotile sperm population is just a consequence of the idea to improve motility by means of the PDE inhibitor pentoxifylline as described above. As pentoxifylline stimulates motility without altering the sperm membrane 238 it appeared as an ideal substance to initiate motility in immotile spermatozoa. This method was successfully used to identify live testicular and epididymal spermatozoa and live births are reported 155156239.
The other option to identify spermatozoa suitable for ICSI is to detect their viability by means of their membrane integrity by the HOS test, which has originally been described by Jeyendran et al. 240. To our knowledge, this method was first proposed by Pike et al. 241. In a small study group of 7 patients these authors showed markedly improved fertilization rates when oocytes were injected with HOS test-positive sperm. Others who also demonstrated significantly higher pregnancy rates eventually confirmed these positive results 242243244245. Eventually, the basic technique has been modified and simplified. All of these modifications resulted in the same promising outcome, significantly elevated fertilization and pregnancy rates 246247248249. Buckett 250 suggested a combined HOS test with the eosin-nigrosin stain as a routine test in the andrological laboratory diagnosis to predict the spermatozoa's fertilizing ability in patients with severe and complete asthenozoospermia.
At this point, however, it is also important to mention the possible consequences of fertilization of oocytes with sperm by ICSI. As sperm motility is also significantly negatively correlated with the production of reactive oxygen species in the spermatozoa themselves and positively correlated with sperm DNA fragmentation 251, the probability to select such DNA-damaged spermatozoa for ICSI is far higher. According to present knowledge, sperm DNA fragmentation might not only cause an impaired embryonic development and early embryonic death 252253254, but also an increased risk of childhood cancer in the offspring 255256. The latter is due to the vulnerability of human sperm DNA during late stages of spermatogenesis and epididymal maturation. At this stage, DNA repair mechanisms have been switched off, resulting in a genetic instability of the male germ cells 257, especially on the Y-chromosome resulting in male-specific cancers 258. Therefore, a careful examination and counselling of the patients seems mandatory, and fertilization with ICSI should not be performed at all cost.
Scavengers for reactive oxygen species (ROS)
In order to maintain cellular polarity and function, spermatozoa contain an extraordinary high amount of polyunsaturated fatty acids, particularly docosahexanoic acid, which has six double bonds per molecule in the plasma membrane. This, in combination with the sperm cell's lack of defence systems, which is due to the obvious deficiency in cytoplasm, and therefore its inability to repair membrane damages effectively renders spermatozoa particularly susceptible to oxidative stress 78. As sperm functions are membrane functions, oxidative damage for any method of assisted reproduction should be reduced to a minimum in order to obtain a maximum of functionally competent male germ cells and to achieve fertilization and pregnancy. Apart from the different sperm separation methods (as discussed above), which also have a significant influence on the ROS production of the spermatozoa, another option to improve sperm functionality and motility is to reduce the detrimental effects of reactive oxygen species on spermatozoa in vitro or in vivo by means of scavenging these highly reactive molecules.
In vivo, spermatozoa depend on scavenging systems provided by the seminal plasma, which is the biological fluid that contains more antioxidant substances than anyone else does. The most important natural antioxidants in seminal plasma seem to be vitamin C and E 259260, superoxide dismutase 261, uric acid 262, glutathione 263 or the polyamine spermine that acts directly as a free radical scavenger 264. Patients with fertility problems show reduced levels of the antioxidative capacity of the seminal plasma 265266267. Thus, in the context of oxidative damage of sperm function not only an excessive ROS production by either leukocytes 268 or the spermatozoa themselves 75269, but also the provision of a sufficient protective antioxidative system in the male genital tract, including the seminal plasma and the female genital tract is of paramount importance. In attempts to tackle the problem of oxidative stress, spermatozoa are either treated with different antioxidants in vitro or the patients are treated with antioxidative drugs.
GLUTATHIONE / N-ACETYL-L-CYSTEINE
As sulfhydryl groups (SH-groups) play an important role in sperm metabolism and the antioxidative defence, and glutathione (γ-glutamyl-cystenyl-glycine) is a natural, highly effective reducing agent, this substance has been tried as a tool to treat male infertility. In a placebo-controlled, double-blind study, in which 20 patients were daily injected with 600 mg glutathione, Lenzi et al. 270 showed a significant increase in sperm motility, percentage of progressive motility and normal sperm morphology. For in vitro treatment of spermatozoa with glutathione during sperm separation, contradictory results have been published. Following swim-up preparation of human spermatozoa in the presence of glutathione Griveau & Le Lannou 271 found an improved acrosome reaction and 24 hours-motility on the same level as for Percoll® gradient centrifugation and suggest that glutathione has a therapeutic potential. In contrast, Donnelly et al. 272 provided data indicating that this drug has no significant effect on progressive motility, neither by itself, nor in combination with hypotaurine. However, the treatment still afforded a significant protection against ROS-induced DNA damage. Unfortunately, so far only very few studies were undertaken in order to treat male infertility using glutathione in vitro or as a treatment for patients. In the human system, a medium based on human tubular fluid supplemented with glucose, taurine and glutathione failed to improve the clinical outcome, fertilization or pregnancy. The morphological quality of the embryos was even lower than in the same medium supplemented with glucose and without phosphate 273. In the porcine and bovine system a positive effect on in vitro maturation of oocytes and blastocyst development, if the spermatozoa were treated with glutathione 274275276 was shown.
Another approach to treat oxidative stress-related male infertility was performed by Oeda et al. 277. These authors used N-acetyl-L-cysteine (ACC), a water-soluble and non-toxic drug that is used in pulmonary diseases because of its strong effect in decreasing the viscosity of sputum by sulfhydryl-disulfide interchange reaction 278. In addition, it was shown that the levels of glutathione in the epithelial lining fluid recovered to normal values after administration of ACC in patients with idiopathic fibrosis of the lung, suggesting that ACC may act as a precursor of glutathione and thus facilitate its biogenesis 279280. In the human ejaculate, ACC revealed a dose- and time-dependent significant reduction of the ROS production 277. In addition, the substance significantly improved motility and did not have adverse effects on viability and acrosome reaction. On the other hand, Hughes et al. 281 demonstrated that the addition of ACC to a sperm separation medium induced sperm DNA damage. In a clinical study, Comhaire et al. 282 treated 27 infertile men orally with ACC and showed a significant reduction of ROS. Moreover, significant increases were found for acrosome reaction, the proportion of polyunsaturated fatty acids of the phospholipids and for the sperm count in oligozoospermic men. However, sperm motility, morphology and the pregnancy rate seemed to be unaltered. Whether or not this is due to the elevated acrosome reaction or the small number of patients included or to a reduced DNA integrity cannot be determined yet. This is a rational approach to treat male factor infertility, which should be followed up in a bigger study.
VITAMINS
Apart from glutathione, vitamins C (ascorbate) and E are natural antioxidants in cells and tissues where ascorbate scavenges highly reactive molecules in the watery phase, i.e. cytoplasm and surrounding liquids, and vitamin E is effective in the lipid phase, i.e. the membranes. In order to improve sperm functions in vitro, culture media were supplemented with ascorbate and vitamin E. While Verma & Kanwar 283 observed a dose-dependent improvement of motility and viability accompanied by a decrease in malondialdehyde production following vitamin E supplementation, Donnelly et al. 284 did not show a beneficial effect of neither ascorbate nor vitamin E on sperm motility. Different motility parameters like curvilinear velocity or linearity were even significantly decreased after the treatment. ROS production was significantly reduced whereas the baseline in DNA damage remained unaltered 285. The simultaneous addition of vitamin C and E to the sperm preparation medium actually induced sperm DNA damage. These negative results of in vitro supplementation with vitamins on human sperm function appear plausible, as the lower levels of ascorbate in the seminal plasma in asthenozoospermic patients 266 can be regarded as a consumption of this antioxidant in vivo. Thus, the oxidative damage of the spermatozoa leading to reduced motility is most probably already set in the testis or epididymis and cannot be repaired by such treatment in vitro.
This consideration is supported by in vivo studies in rabbits, boars and in the human, where vitamin E supplementation resulted in improved sperm functions and a reduced production of free radicals 282286287288. However, there are also negative reports on the effect of vitamin supplementation. In the human, Rolf et al. 289 did not find changes in semen parameters of 31 asthenozoospermic and moderate oligoasthenozoospermic patients treated with high doses of vitamin C and E in a randomized, placebo-controlled, double-blind study. Likewise, no positive effects on semen volume, pH or sperm motility were observed following a dietary vitamin E administration in the cock 290. The reproductive performance of the treated cocks was even negatively influenced. These contradictory results show that neither the exact mechanism and site of action, nor the indication for a successful antioxidative treatment of patients are clear. In addition, the action of genital tract inflammations has not yet been taken into consideration. As leukocytes produce enormous amounts of free radicals 32 and their specific impact on sperm function remains to be clarified, more research must be carried out in order to obtain a rational antioxidative therapy for male factor patients.
PENTOXIFYLLINE
Besides the effects of pentoxifylline on sperm motility and acrosome reaction discussed above, this PDE inhibitor has also effects on ROS. It has repeatedly been shown that pentoxifylline significantly reduces the superoxide release of human spermatozoa following phorbol myristate acetate stimulation 291292. This effect is possibly due to the reduction of the formation of endoperoxides as a consequence of the elevated cAMP levels that inhibit the cyclo-oxygenase within the arachidonic pathway 292. Contradictory results, however, were obtained regarding lipid peroxidation. While Gavella & Lipovac 293 found elevated levels of malondialdehyde after pentoxifylline treatment and rather warned of its use in assisted reproduction, McKinney et al. 294 could not confirm these findings. In a comparative study, Okada et al. 295 confirmed the ROS scavenging and motility stimulating effect of pentoxifylline in vitro in 15 patients and 18 controls, respectively. However, in vivo pentoxifylline at low dosages (300 mg per day) failed to decrease ROS generation and to increase motility. On the other hand, at high dosages (1,200 mg per day), motility and beat cross frequency were increased but the drug still did not have a beneficial effect on sperm fertilizing ability.
Reduction of visco-elasticity of the ejaculate
In most mammals, semen coagulates shortly after ejaculation and liquefies again later on. In the human, liquefaction takes place within 5–20 minutes after ejaculation. While so-called coagulation proteins like semenogelin or fibronektin, which derive from the seminal vesicle, promote coagulation, seminolysis is caused by a prostate-derived serine-proteinase, prostate-specific antigen (PSA) 296. However, in some cases viscosity of the semen remains high and can be a cause for male infertility. It is also important to differentiate between ejaculates that have an excessive viscosity and such ejaculates that failed to liquefy 107. As highly viscous semen can reduce sperm motility thus being a cause for male infertility, it is necessary in the clinical set-up of assisted reproduction either to reduce semen viscosity or to liquefy the ejaculate artificially. For this, a few methods are available. Mixing the semen with medium is the easiest way but will not work sufficiently in cases of excessive semen viscosity. Another method that was suggested in the past is to force the viscous seminal fluid through a narrow-gauge needle. However, considering the severe damage that this method causes to the spermatozoa including immotility, it cannot be recommended at all. It is therefore rather advisable to liquefy such ejaculates enzymatically by using a 0.2% solution of α-amylase. Several other enzymes like α-chymotrypsin, lysozyme or hyaluronidase have also been described for this purpose. Recently, a special trypsin-based dissolving solution, SpermSolute, has been reported 297. However, as these enzymes can also damage spermatozoa, special care should be taken to remove the enzymes by washing procedures as soon as liquefaction is completed.