Reproductive Biology and Endocrinology BioMed Central Review Open Access Sperm preparation for ART Ralf R Henkel* and Wolf-Bernhard Schill Address: Department of Dermatology and Andrology, Justus Liebig University, Giessen, Gaffkystr. 14, Germany Email: RalfRHenkel*[email protected]; Wolf-BernhardSchill-wolf-bernhard.schill@derma.med.uni-giessen.de * Corresponding author Published: 14 November 2003 Received: 11 July 2003 Accepted: 14 November 2003 Reproductive Biology and Endocrinology 2003, 1:108 This article is available from: http://www.rbej.com/content/1/1/108 © 2003 Henkel and Schill; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Abstract The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm. Page 1 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 Introduction range of different sperm separation methods. Following Since the birth of Louise Brown on 25 July 1978 and the the development of the classical swim-up method by subsequent onset of assisted reproduction in the human, Mahadevan & Baker [4], more complicated techniques scientists and clinicians were more and more urged to were developed to increase the number of motile sperma- improve sperm separation techniques as the percentage of tozoa even in severe andrological cases. On principle, andrological cases increased rapidly. While the first in vitro these techniques can be differentiated in migration, den- fertilization (IVF) cases, including that of Louise Brown, sity gradient centrifugation and filtration techniques. For were performed to treat tubal infertility, the increasing all migration methods, the self-propelled movement of number of men showing poor semen quality prompted spermatozoa is an essential prerequisite, while for density the development of a wide array of different laboratory gradient centrifugation and filtration techniques the techniques focusing on the selection and enrichment of methodology is based on a combination of the sperm motile and functionally competent spermatozoa from the cells' motility and their retention at phase borders and ejaculate. The first sperm separation methods available adherence to filtration matrices, respectively. The migra- only comprised of one or two washing procedures with tion techniques can again be subdivided into swim-up, subsequent resuspension of the male germ cells [1-3]. under-lay and migration-sedimentation methods [8,9]. Mahadevan and Baker [4] then described a single wash For density gradient centrifugation, separation media like followed by a swim-up procedure from the cell pellet. Fol- Ficoll® [10], Nycodenz [11] and Percoll® [12,13] including lowing these first reports on human sperm separation, the products (IxaPrep®, PureSperm®, Isolate®, SilSelect®) more sophisticated methods were developed to obtain have recently been introduced to replace Percoll® [14,15]. sufficient amounts of motile, functionally competent The filtration methods like glass wool filtration [16,17] spermatozoa for IVF. Eventually, methods were developed and filtration of spermatozoa on Sephadex beads [18] and that improved sperm functions like motility, protected membranes [19] are alternative techniques. sperm functions and/or reduced detrimental effects from the environmental milieu like reactive oxygen species. The ideal sperm separation technique should (i) be quick, easy and cost-effective, (ii) isolate as much motile sperma- In this paper, we aimed at giving an update on the main tozoa as possible, (iii) not cause sperm damage or non- sperm separation methods including their implications physiological alterations of the separated sperm cells, (iv) and importance for modern assisted reproductive tech- eliminate dead spermatozoa and other cells, including nologies as well as an overview on different in vitro treat- leukocytes and bacteria, (v) eliminate toxic or bioactive ments of spermatozoa to improve their functional substances like decapacitation factors or reactive oxygen competence and to reduce detrimental effects. species (ROS), and (vi) allow processing of larger volumes of ejaculates. Since none of the methods available meets Sperm separation techniques all these requirements, a variety of sperm separation tech- Under in vivo conditions, potentially fertile spermatozoa niques is mandatory in clinical practice to obtain an opti- are separated from immotile spermatozoa, debris and mal yield of functionally competent spermatozoa for seminal plasma in the female genital tract by active migra- insemination purposes. Depending on the ejaculate qual- tion through the cervical mucus [5]. During this process, ity, these methods have different efficiency and areas of not only progressively motile spermatozoa are selected, use. In the conventional swim-up technique, functional but male germ cells also undergo physiological changes spermatozoa can come into close cell-to-cell contact with called capacitation, which are fundamental prerequisites defective sperm or leukocytes by centrifugation, thus caus- for the sperm's functional competence with regard to ing massive oxidative damages of the sperm plasma mem- acrosome reaction [6,7]. brane by ROS and consequently of sperm functions [20]. Therefore, the quality of the ejaculates has direct conse- The introduction of assisted reproduction, especially of quences on the choice of a sperm separation method. IVF, during the 1980's, led to the development of a wide Table 1: 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 Page 2 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 With regard to the possible risk of a seroconversion in percentage of normally chromatin-condensed spermato- women and in the offspring after performing ART with zoa has been reported after the swim-up procedure [31]. spermatozoa from a HIV-positive man initial concerns Another major disadvantage of this technique is the fact arose as HIV viruses reportedly can bind to and penetrate that for its use spermatozoa are pelleted, thus coming into into spermatozoa [21,22]. Later research, however, close cell-to-cell contact with each other, cell debris and showed that HIV-1 genomes found to an extend of 18% leukocytes, which are known to produce very high levels in seminal cells samples decreased to undetectable levels of reactive oxygen species (ROS) [32]. Due to the extraor- following a combined density gradient centrifugation dinary high amount of poly-unsaturated fatty acids in the with a subsequent swim-up [23]. No seroconversion of sperm's plasma membranes [33], these ROS cause lipid the virus could be observed after fertilization of oocytes by peroxidation and therefore a dramatic decrease in sperm IVF-ICSI [24-26]. Other recent research [27] showed that functions, including motility [34]. Overall, although HIV RNA and DNA could be detected in separated sper- many men's spermatozoa may not be impaired to the matozoa even in treated patients. Thus, viral validation of extent of inhibiting fertilization, some couples' chances of separated spermatozoa is necessary and should be per- successful IVF will certainly be compromised. It is there- formed even in treated patients. Additionally, only HIV- fore not reasonable to continue and to use a technique, tested maternal serum or commercially available serum such as swim-up from pelleted semen with the inherent albumin, which is HIV-free due to its processing, should potential to cause irrevocable damage to spermatozoa be used as protein supplement for culture media. prejudicial to a desired functional endpoint. Eventually, this knowledge led to the development of other more gen- In order to give an overview, only the four most common tle sperm separation methods that also allow a higher techniques classical swim-up, migration-sedimentation, recovery of motile and functional spermatozoa. The density gradient centrifugation and glass wool filtration advantages and disadvantages of the conventional swim- are discussed. up are summarized in table 1. Swim-up procedure An attempt to overcome at least the problems caused by Apart from a simple wash and subsequent resuspension of ROS, the "swim-up" can be performed directly from the the male germ cells, the swim-up from a washed pellet is liquefied semen. During this procedure, several aliquots the oldest and most commonly used sperm separation of liquefied semen are taken from a sample and placed in method. Originally described by Mahadevan and Baker tubes underneath an overlay of culture medium. Round- [4], this method is still used largely in IVF laboratories bottom tubes or 4-well dishes should be used to optimize around the world. Although its use among the male factor the surface area of the interface between the semen layer infertility group is very limited, the swim-up is still the and the culture medium. The tubes may also be prepared standard technique for patients with normozoospermia by gently layering culture medium over the liquefied and female infertility. Excellent fertilization rates were semen. The placing of semen underneath the culture reported when these sperm preparations were used to medium, however, provides a much cleaner interface inseminate human oocytes in vitro. However, as the indi- zone. A maximum recovery is obtained by using multiple cations for IVF were expanded beyond simple tubal factor tubes with small volumes of semen per tube, thus maxi- cases to couples with idiopathic infertility and, ultimately, mizing the combined total interface area between semen to male factor cases, the problem of fertilization failure and culture medium. Mortimer [35] suggested the use of appeared [28-30]. 250 µl semen and 500 to 600 µl culture medium per tube. After the incubation period, which is typically between 30 The methodology of this conventional swim-up is based and 60 minutes, at 37°C, most of the upper culture on the active movement of spermatozoa from the pre- medium layer is removed. This should be done with cau- washed cell pellet into an overlaying medium. Typically, tion, working from the upper meniscus downwards, using the incubation time is 60 minutes. This technique is dis- a sterile pipette. Typically, 75 or 80% of the culture tinguished by a very high percentage (>90%) of motile medium layer are removed and eventually combined, sperm, preferred enrichment of morphologically normal taking great care not to aspirate directly from the interface spermatozoa as well as the absence of other cells and region. This procedure will also increase the total number debris. Considering that the efficiency of the technique is of recovered spermatozoa, which can then also be used for based on the surface of the cell pellet and the initial sperm ICSI [36]. motility in the ejaculate, the yield of motile spermatozoa is limited. Many layers of cells in the pellet may cause Other swim-up methods include the swim-up of sperma- potentially motile spermatozoa in the lower levels of the tozoa in a specially supplemented medium. Such sub- pellet never to reach the interface with the culture stances can be SpermSelect™, which is a highly purified medium layer. In addition, a significant decrease in the preparation of hyaluronic acid (Pharmacia, Uppsala, Page 3 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 Table 2: 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 Table 3: Advantages and disadvantages of density gradient centrifugation. Advantages Disadvantages - usually clean fraction of highly motile spermatozoa - production of good interphases between the different media is a bit more time-consuming - spermatozoa from ejaculates with a very low sperm density can be - a bit more expensive separated - good yield - potential risk of endotoxins - leukocytes are eliminated to a large extent - Percoll® may no longer be used IVF/ICSI - reactive oxygen species are significantly reduced Sweden) with an average molecular weight of 3,000 kDa medium and subsequently sediment in that inner cone that was used at a final concentration of 1 mg/ml in cul- within an hour's time. Thus, this method is a highly gentle ture medium. Compared with the traditional swim-up technique, especially if compared with methods that from a washed pellet [37], a swim-up directly from semen require centrifugation steps before the sperm separation into a hyaluronic acid solution gave a significantly higher like the conventional swim-up. In the original version, a percentage of motile spermatozoa and, ultimately, the fraction of highly motile and functionally competent achievement of a higher pregnancy rate in a clinical IVF spermatozoa can be obtained. Unfortunately, the yield is program [38]. However, highly purified hyaluronic acid is very low and therefore the original method did not find expensive and it has been shown to increase the calcium- wide acceptance for IVF. The advantages and disadvan- influx into spermatozoa and therefore induce acrosome tages of this method are summarized in table 2. Recently, reaction [39]. Elevated local concentrations of hyaluronic Zavos et al. [9] proposed the use of a multi-chamber tube acid in the cumulus oophorus have been shown to con- to retrieve functional spermatozoa for assisted reproduc- tribute to the acrosome reaction [40]. Thus, it seems rather tive techniques by means of a swim-up and sedimentation questionable whether this substance is favourable for IVF. method. The assessment of its usefulness for IVF/ICSI, In addition, whether these improved results in sperm however, is still to be awaited. motility were specifically due to the use of the hyaluronate or to the use of a method, which did not involve the initial Sánchez et al. [44] showed that after concentration of pelletting of unselected spermatozoa, has not been ascer- sperm cells in the ejaculate, even in cases with severe tained. On the other hand, however, hyaluronic acid has oligo- and/or asthenozoospermia a sufficient number of been regarded as an effective alternative to test sperm pen- motile spermatozoa can be isolated for intracytoplasmic etration into human cervical mucus [41-43]. sperm injection (ICSI) after 2–3 hours of incubation. Compared with the density gradient centrifugation, these Migration-sedimentation authors also demonstrated significantly better results for A more sophisticated sperm separation technique is progressive motility, normal sperm morphology, chroma- migration-sedimentation, which was developed by Tea et tin condensation and reduction of the percentage of dead al. [8]. Principally, this method is a swim-up technique spermatozoa as determined by the eosin test. In addition, combined with a sedimentation step. Special glass or plas- since spermatozoa isolated by this modified migration- tic tubes with an inner cone are used. In contrast to the sedimentation technique stick markedly less to the glass conventional swim-up procedure, spermatozoa swim up surface of the injection pipettes, this method has even an directly from liquefied semen into the supernatant advantage over the density gradient centrifugation, which Page 4 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 is normally used for these cases. In this regard, the side ent was found to produce populations of highly motile migration technique that was recently proposed by Hint- spermatozoa with better yields and survival than either ing and Lunardhi [45] is another interesting approach to swim-up or Percoll® gradients from oligozoospermic and obtain motile spermatozoa from very poor quality semen asthenozoospermic semen samples [11,59]. Compared for ICSI as it also yielded better sperm quality. with the conventional swim-up procedure from a pelleted sperm population, the use of Nycodenz also seems to be Density gradient centrifugation superior regarding sperm penetration into zona-free ham- The typical methodology for the density gradient centrifu- ster eggs [60]. Thus, this technique has clearly great poten- gation comprised continuous [46] or discontinuous gra- tial in the preparation of motile spermatozoa from poor dients [47]. With continuous gradients, there is a gradual quality semen for IVF use and warrants further increase in density from the top of the gradient to its bot- investigation. tom, whereas the layers of a discontinuous gradient show clear boundaries between each other. The ejaculate is Other replacement products for Percoll® that were intro- placed on top of the density media with higher density duced into the market from the mid nineties and more and is then centrifuged for 15–30 minutes. During this commonly used in assisted reproduction are IxaPrep® procedure, all cells reach the semen sediment. However, (MediCult, Copenhagen, Denmark), SilSelect® (FertiPro highly motile spermatozoa move actively in the direction N.V., Beernem, Belgium), PureSperm® (NidaCon Labora- of the sedimentation gradient and can therefore penetrate tories AB, Gothenburg, Sweden) or ISolate® (Irvine Scien- the boundary quicker than poorly motile or immotile tific, Santa Ana, CA, USA). In contrast to Percoll®, which is cells, thus, highly motile sperm cells are enriched in the a PVP-coated silica that can have deleterious effects on soft pellet at the bottom. sperm membranes [56], all these replacement products contain silane-coated silica particles, are adjusted for the A wide variety of methods using the principle of density osmolarity with polysucrose and have very low toxicity. gradient centrifugation to fractionate subpopulations of All these replacement products are non-irritating and are spermatozoa has been described in the literature. Ficoll® approved for human in vivo use. The results of sperm prep- has initially been used as gradient material for preparing aration using these new products compared with Percoll® spermatozoa [48], but by far the most widely used sub- regarding recovery rate, motility, viability, normal sperm stance for all methods of assisted reproduction (IUI, GIFT, morphology and velocity parameters like VAP or VCL vary IVF, ICSI, etc.) have been the polyvinylpyrrolidone (PVP)- considerably among different working groups. While coated silica particles Percoll®. Normal sperm function in Claassens et al. [61] and Söderlund and Lundin [62] did terms of sperm fertilizing ability as assessed in the zona not find differences in the recovery rate between Percoll® pellucida-free hamster egg penetration test [20,49], as and PureSperm®, Chen and Bongso [63], depending on well as in human IVF [13] and ICSI [50] was observed. In the number of layers included for the density gradient, October 1996, Percoll® has been withdrawn from the mar- reported significantly higher values for PureSperm®. For ket for clinical use in assisted reproduction [51]. This was IxaPrep® it is even more confusing because Yang et al. [64] because of the risk of contaminations with endotoxins found no difference to Percoll®, while Makkar et al. [14] [52-54], possible membrane alteration [55,56] and found the replacement substance more effective. On the inflammatory responses that could be induced by the other hand, McCann and Chantler [65] as well as Ding et insemination of sperm populations contaminated with al. [66] found Percoll® superior. These authors attribute Percoll®. In addition, Percoll® adheres to the sperm mem- the better sperm quality obtained after the IxaPrep® prep- branes [57] and might alter them by removing coating aration to a significantly decreased production of nitric envelopes [58]. Therefore, intensive washing of the sper- oxide, which is regarded as sperm toxicant that reduces matozoa after sperm separation with Percoll® was recom- motility [67]. This could be due to an activation of guan- mended [55]. This requires additional centrifugation and ylyl cyclase, thus increasing cGMP production, which can again be detrimental to the spermatozoa because of inhibits sperm motility [68]. On the other hand, nitric the action of reactive oxygen species [20]. oxide is also known to be a physiologic mediator for vasodilatation, immunosuppression, neurotransmission Another commercial product known as Nycodenz (Nye- and cytotoxicity [69-72]. gaard & Co., Oslo, Norway) was also used as a density gra- dient material for preparing human spermatozoa. Regarding the other parameters such as motility, viability, Nycodenz is the same molecule, iohexol, as used in the X- normal sperm morphology or velocity parameters like ray contrast medium Omni-paque. Studies revealed a low VAP, the data currently available also vary considerably incidence of adverse reactions during angiography [11]. among different working groups. The reason for this can Both continuous and discontinuous Nycodenz gradients be attributed to the different conditions of the sperm sep- were evaluated, of which a four-layer discontinuous gradi- aration, e.g. volume of semen to be separated, g-force, Page 5 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 Table 4: 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 - remnants of debris are still present separated - good yield - leukocytes are eliminated to a large extent - reactive oxygen species are significantly reduced centrifugation time or the number of layers of the gradi- step will be necessary to remove the seminal plasma. This ent, and reflect the important role of the methodology. is an important aspect as this procedure reduces cellular Moreover, this also shows that these data cannot be damage by reactive oxygen species. The advantages and directly compared. Overall, the Percoll®-replacement disadvantages of this method are summarized in table 4. products are good and reasonable alternatives, and this not only for the fact that Percoll® is no longer allowed to By means of glass wool filtration, it is even possible to pre- be used for clinical purposes in assisted reproduction. pare motile spermatozoa from patients with retrograde ejaculation (Henkel et al., unpublished). In these cases, Glass wool filtration the procedure includes adjustment of the osmolarity of During glass wool filtration, which has already been the patient's urine to values of about 350 mOsmol/kg by described by Paulson & Polakoski in 1977 [17], motile drinking water. Prior to the ejaculation, the patients are spermatozoa are separated from immotile sperm cells by requested to urinate most of the urine in the bladder. The means of densely packed glass wool fibres. The principle small amount of antegrade-produced ejaculate is col- of this sperm separation technique lies in both the self- lected in a plastic beaker, while the retrograde fraction of propelled movement of the spermatozoa and the filtra- the ejaculate needs to be urinated immediately into a jar tion effect of the glass wool. The success of this method is with 50 ml culture medium containing human serum directly linked to the kind of glass wool used [73]. Thus, albumin to dilute the urine, Finally, the urine/medium factors like the chemical nature of the glass (i.e. borate mixture has to be centrifuged, resuspended in 3 to 4 ml of glass, silicate glass or quartz glass), the surface structure fresh medium and filtrated on the glass wool column. As and charge of the glass wool, thickness of the glass wool constituents of the urine can damage the spermatozoa, a fibres or the pore size of the filter have to be taken into speedy work-up of such ejaculates is mandatory. consideration. In clinical practice, the glass wool, code number 112, from Manville Fiber Glass Corp. (Denver, In addition to the separation of spermatozoa, glass wool CO, USA) or SpermFertil® columns from Mello filtration has been shown to eliminate leukocytes to an (Holzhausen, Germany) have been tested extensively. extent of up to 90% [75]. Since leukocytes are frequent Potential risks of the technique such as damages of the even in normal ejaculates [76] and produce 100-times spermatozoa or the occurrence of glass wool fragments in more ROS than spermatozoa [32], this effect significantly the filtrate essentially depend on the kind of glass wool contributes to a reduction of free radicals in the ejaculate used and on the intensity of the washing prior to the [73,75]. This is of paramount importance for the func- filtration. tionality of spermatozoa because the male germ cells are particularly susceptible to oxidation by ROS because of Compared with the swim-up or migration-sedimentation, their extraordinary high content of polyunsaturated fatty glass wool filtration, just as density gradient centrifuga- acids in their plasma membrane [33,77,78]. tion, is a technique that uses the whole volume of the ejac- ulate and therefore yields a significantly higher total Another clinically interesting aspect related to glass wool number of motile spermatozoa [31,74]. Thus, it can also filtration is chromatin condensation, which has repeat- be used for patients with oligo- and/or asthenozoosper- edly been shown to be predictive of fertilization in vitro mia [74]. Like density gradient centrifugation, glass wool [79-81]. Glass wool filtration [31] like the density gradi- filtration also provides the advantage that the sperm sep- ent centrifugation with PureSperm® [82] or the migration- aration can directly be performed from the ejaculate. Only sedimentation technique [44] significantly selects nor- after the separation of the functional spermatozoa from mally chromatin-condensed spermatozoa, while conven- the immotile ones, leukocytes and debris, a centrifugation tional swim-up or Percoll®-centrifugation decrease this Page 6 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 sperm parameter. As human sperm chromatin condensa- filter. These filters are unusual because their pores are tion follows a seasonal rhythm, which even shows a shift cylindrical and at right angles to the plane of the mem- of about half a year on the southern hemisphere [83], this brane [90]. The spermatozoa, therefore, have straight might have a clinical impact on the results in IVF. Should channels to swim through the membrane. Unfortunately, a patient be examined in winter when the quality of sperm these membranes had a very low ratio of the total cross- chromatin condensation is high [83] and referred to IVF sectional area of the pores to the overall membrane area. in summer when the percentage of normally chromatin- Consequently, the yield is extremely low. Primarily, this condensed spermatozoa is significantly lower, IVF for this method was used for testing the motility of sperm popu- patient might fail. Thus, for these patients a sperm separa- lations treated with various pharmacological agents, but tion by means of glass wool filtration, PureSperm® or not as a preparation method for assisted reproduction migration-sedimentation might be beneficial. [91]. Glass beads Another approach of separating viable human spermato- This method has been used for the preparation of hamster zoa by means of membranes was undertaken by Agarwal spermatozoa for in vitro capacitation [84] and resulted in et al. [19] using a membrane which has been developed an efficient, high yield selection of motile human sperma- for selective removal of leucocytes (L4 membrane). tozoa from semen [85]. However, there were concerns Besides a significant increase of motility, ejaculates fil- regarding the possible spill over of beads into the insemi- tered through this membrane have been shown to contain nation medium. As a result, the use of glass beads for fewer leukocytes. This fact is, of course, of importance in effective sperm preparation for assisted reproduction has those cases that have increased numbers of leucocytes in not widely been accepted. the ejaculate as a result of infections. Moreover, this mem- brane seems to be selective for spermatozoa with normal Sephadex columns membrane integrity [92,93] and sperm producing low In the early nineties sperm separation by means of Sepha- amounts of reactive oxygen species [94]. However, despite dex beads emerged [18] and a commercial sperm separa- these advantages of the membrane it has never come into tion kit based on this principle (SpermPrep®) has become practical clinical use for human assisted reproduction. available. Compared to migration-sedimentation and swim-up from pelleted semen it produced significantly Improvement of sperm concentration in the higher yields [86]. Moreover, morphologically normal fertilization well sperm cells could be enriched in the filtrate after Sperm- Apart from the different sperm separation techniques to Prep® separation as well as significantly higher pregnancy increase the number of functional spermatozoa for rates for intrauterine insemination as compared with the assisted reproductive techniques discussed above, trials conventional swim-up method [87]. In a comparison have been made to improve fertilization rates by modify- between SpermPrep® method and Percoll® centrifugation, ing the conditions of the co-culture of oocytes with sper- Percoll® separated spermatozoa showed a significantly matozoa. Micro-insemination techniques have especially higher percentage of normally chromatin-condensed and been developed for patients with sperm counts or asthe- morphologically normal spermatozoa [88]. However, the nozoospermia. One of the first who used such a method fertilization rates reported by these authors were similar. for assisted reproduction were Ranoux and Seibel [95]. López et al. [89] used a prepacked PD-10 column contain- These authors used a microvolume straw to co-incubate ing Sephadex G-25 particles (Pharmacia Biotechnology, spermatozoa with the oocytes and incubated this straw Uppsala, Sweden), which is normally used to desalt pro- intravaginally, thus they called the technique intravaginal teins in solutions, to separate human spermatozoa and culture (IVC). Giorgetti et al. [96] proposed a swim-across compared the results with the SpermPrep® method and technique in a medium that contains human follicular Percoll® centrifugation. The PD-10 column and density fluid and found a significantly improved fertilization rate gradient centrifugation in Percoll® yielded a comparable for patients with motile sperm counts less than 1 × 106 number of spermatozoa and showed similar percentages spermatozoa/ml of semen. Fishel et al. [97] reported crit- of morphologically normal spermatozoa after sperm sep- ically on the microdrop IVF and preferred microinjection. aration. On the contrary, the SpermPrep® method resulted On the other hand, Svalander et al. [98] presented encour- in significantly lower values of sperm count and aging results with a fertilization rate of 49.3% and a baby- morphology. take-home rate of between 20–24.7%. Although this is lower than the baby-take-home rate reported for conven- Transmembrane migration tional IVF (27–31.7%), these authors recommended the Another alternative sperm separation technique that was technique for moderate male factor infertility. also developed in the late eighties is migration/filtration of motile spermatozoa through a Nuclepore membrane Page 7 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 This success of the technique may be dependent on the In vitro treatment of spermatozoa volume of the microdrop as well as on the sperm concen- An alternative and/or complementary approach to sperm tration. In this regard, Özgür et al. [99] developed a pre- preparation for ART in vitro by means of the above dis- dictive model to calculate the therapeutically optimal cussed sperm separation methods is the idea to treat the sperm concentration for a defined microvolume. This spermatozoa in vitro in order to improve their functional- might be explained by data of Özgür and Franken [100] ity, i.e. motility, or to supply a protective environment who showed that sperm-zona binding is dependent on with the purpose to maintain or improve their functional the sperm-zona collision rates, which is dependent on the capacity for successful fertilization. Many substances size of the microdrops itself. However, as the results of the including serum, peritoneal fluid and follicular fluid or microdrop IVF were conflicting, and successes sporadic, other chemically defined pharmacological substances like intracytoplasmic sperm injection (ICSI) has become the progesterone, adenosine analogues or methylxanthins method of choice in the treatment of severe male factor have been proposed to stimulate human sperm functions. infertility. The methodologies addressing the different aspects, motility and sperm functions, ROS, and the reduction of Immunological infertility and ART the visco-elastic properties of the semen are discussed Immunologic infertility due to sperm autoimmunity below. afflicts 5–10% of infertile couples [101]. Normally, male germ cells, which express surface antigens that are not present on somatic and pre-meiotic cells, are shielded A B from immuno-competent cells in the body by the blood- testis barrier. This prevents an immunologic reaction. O H O CH3 Htiso wbeavrerire, ri nb thy e ienvfeenctti oofn as ,d iisninjutreigersa tioorn douf teh et ob losoudrg-itceas-l H N N CH3 N N treatments, these actually "strange" surface antigens can O N N O N N be exposed to the immune system. As a result, the body produces antibodies against its own spermatozoa. On the H CH 3 other hand, women can also produce anti sperm-antibod- ies. One possible target for such antibodies is the human sperm protein rSMP-B [102]. These antibodies can coagu- late and immobilize the spermatozoa or can even be cyto- C toxic [103] and eventually lead to infertility as well as reduced fertilization rates in assisted reproduction [104]. O O CH3 However, contradictory results that the presence of sperm autoantibodies in female sera used for media in IVF does CH3CCH2CH2CH2CH2 N N not affect the IVF result are also reported [105]. O N N In the past, it was thought that anti sperm-antibodies CH 3 could be eliminated just by washing them off. However, D as normal antigen-antibody reactions usually have affinity constants between 107 and 109 l/mol, a simple washing NH 2 step will not suffice for removing these antibodies from N the sperm surface. As in men with sperm autoantibodies N not all spermatozoa are affected by the antibodies, Hint- N N ing et al. [106] could significantly reduce the anti sperm- O antibodies as tested with the mixed antiglobulin reaction HOCH2 test (MAR test) by using a swim-up technique with fetal cord serum. The cumulative pregnancy rates of treatments HO H for intrauterine insemination (IUI) in cases of immuno- logical infertility are reported between less than 10% MlFiniogel ue(rCceu)l a1arn dst r2u-dcteuorxeysa odfe nxoansitnhein (eD ()A), caffeine (B), pentoxifyl- [107] and up to 25% [108]. Today, the method of choice Molecular structures of xanthine (A), caffeine (B), pentoxifyl- to treat severe cases of immunological infertility is IVF or line (C) and 2-deoxyadenosine (D). even ICSI where pregnancy rate of 30–40% per cycle can be achieved. Page 8 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 Improvement of motility and sperm functions and abnormal sperm morphology in a total of 546 men. CAFFEINE In combination with smoking as further risk factor, sperm The use of xanthine (Fig. 1A) derivatives like the methylx- motility and viability decreased significantly. Klonoff- anthines caffeine (1,3,7-trimethyl-2,6-dioxopurine) (Fig. Cohen et al. [120] confirmed the embryotoxic effect of 1B) and pentoxifylline (Fig. 1C) for the pharmacological caffeine causing delayed conception [122] or spontaneous stimulation of sperm functions, especially for motility, is abortions [123] and concluded that the caffeine well known. Garbers et al. [109] already suggested stimu- consumption should be minimized prior to and during lation of human sperm motility by caffeine. This sub- an assisted reproduction treatment. stance is an inhibitor of the phosphodiesterase that leads to increased cellular levels of cAMP. Considering that a PENTOXIFYLLINE defective energy metabolism of the spermatozoa is also a Pentoxifylline (Fig. 1C) is another methylxanthine deriv- potential cause of male infertility, the use of motility stim- ative, which is also, like caffeine, a non-specific inhibitor ulating substances to improve sperm motility and there- of phosphodiesterase. In contrast to caffeine, the Food fore fertilization rates appeared reasonable. and Drug Administration (FDA) approved pentoxifylline for the administration to humans. The drug is used for sys- Human spermatozoa can obtain energy from both glyco- temic treatments of patients with cardio-vascular diseases lysis and mitochondrial oxidative phosphorylation [110]. (Trental® or Torental®). In addition, since its water solubil- Thus, motility parameters such as mode of progression, ity is higher than that of caffeine, this increases its usabil- lateral head displacement or velocity of fresh and cryopre- ity [124]. served spermatozoa significantly increased after stimula- tion with caffeine due to augmented glycolysis and The beneficial effect of pentoxifylline on sperm motility fructolysis [111-113]. A small study including 6 patients and motion characteristics like sperm velocity or hyperac- by Serres et al. [114] revealed that the stimulatory effect is tivity has repeatedly been described for both fresh most obvious in patients with asthenozoospermia. How- [112,125-129] and cryopreserved spermatozoa [128,130- ever, early reports documented not only a stimulating 132]. The results on the stimulation of sperm motility are effect of caffeine, but also a time- and concentration- conflicting. Yovich et al. [133], Rees et al. [112] and Lewis dependent detrimental effect on the sperm plasma mem- et al. [134] found no effect in normozoospermic patients, brane [115,116]. Moreover, spermatozoa pre-incubated while others [133-136] observed a significant increase in with caffeine showed a significant decrease in the percent- motility and the number of progressively motile sperma- age of penetrated zona-free hamster oocytes [117]. The tozoa in patients with asthenozoospermia. This stimula- only study available [118] that investigated the effect of tory effect can clearly be attributed to the increased caffeine in assisted reproduction using 42 oocytes from 21 intracellular levels of cAMP. In the absence of the drug, women showed an increase in sperm motility but a signif- the cAMP content remained unchanged and correlated icant decrease in the fertilization rates and embryo only with hypermotility and the amplitude of the lateral development. head displacement of the spermatozoa [137]. Cyclic AMP, in turn, is believed to stimulate a cAMP-dependent kinase Direct incubation of mouse oocytes and embryos resulted [138], which itself induces sperm tail protein phosphor- in a time- and concentration-dependent artificial parthe- ylation [139] with subsequent increase in sperm motility nogenic activation of the oocytes and a significant reduc- [140]. tion of embryo cleavage rates [119]. As these data were more disappointing than stimulating for the clinical use Apart from the effects on sperm motility, pentoxifylline is of caffeine in assisted reproduction, it was further only also reported to augment acrosome reaction [141]. Nassar used for research purposes. A very recent prospective study et al. [142] demonstrated that this induction is not due to by Klonoff-Cohen et al. [120] investigated the daily caf- a Ca2+-influx in the sperm cell, which is regarded to stim- feine consumption of 221 patients and its effects on the ulate acrosome reaction [143]. The intracellular [Ca2+] i outcome of IVF/GIFT treatments. According to these even decreased following the pentoxifylline treatment. authors, failed pregnancies were significantly associated Since cAMP is intimately involved as a second-messenger with the female's caffeine consumption. In addition, ges- in the induction of acrosome reaction [144], an unspecific tational age at delivery decreased significantly in women inhibition of phosphodiesterases by this methylxanthine who consumed more than 50 mg/day. Male caffeine con- will increase the intracellular cAMP levels and therefore sumption appeared to be a significant risk factor for mul- induce acrosome reaction. In addition, this drug also tiple gestations but had no effect on sperm count, motility improves the spermatozoa's zona pellucida binding abil- or normal sperm morphology. An earlier study [121], ity [139,145,146]. However, it appeared that this binding however, demonstrated a positive association between improvement to the zona pellucida is rather a result of the coffee consumption and sperm concentration, motility increase in the sperm velocity parameters straight-line Page 9 of 22 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/108 velocity (VSL) and average path velocity (VAP) as these unspecific inhibition of the PDE's will obviously result in parameters are indicators of progressive motility. There- both, stimulation of motility and acrosome reaction. fore, these spermatozoa represent a sperm population Depending on the conditions and most importantly on that did not initiate acrosome reaction with its character- the time of stimulation and the concentration of pentoxi- istic change in movement characteristics yet [147]. fylline in the medium, over-stimulation will definitely result in a too early acrosome reaction. Hence, over-stim- The benefits of a treatment with pentoxifylline prompted ulated spermatozoa for an IUI or IVF treatment will not its use in assisted reproduction programs. However, the fertilize oocytes because they are no longer able to bind to results reported in the literature are rather conflicting. the zona pellucida. This dilemma might be overcome by While Tasdemir et al. [148] and Tarlatzis et al. [149] the use of a non-embryotoxic PDE-4 inhibitor to stimu- found an improvement in the IVF rate and saw a promis- late sperm motility only. Unfortunately, to our knowl- ing development in small studies comprising 51 and 43 edge, no further progress has been made in this regard. For patients, respectively. Others did not find differences ICSI, this problem is not relevant as spermatozoa bypass between the control and the treatment group in IVF and all physiological barriers because they are directly injected intrauterine insemination [150-152]. Thus, it was con- into the oocytes. cluded that pentoxifylline should not be used indiscrimi- nately [153]. On the other hand, pentoxifylline has been 2-DEOXYADENOSINE successfully used to increase fertilization rates in bovine in 2-Deoxyadenosine (2-DA) (Fig. 1D) is an adenosine vitro fertilization [154] and as pre-treatment to stimulate derivative that is not a phosphodiesterase inhibitor like epididymal and testicular sperm motility for ICSI pentoxifylline or caffeine. Like these substances, 2-DA is [155,156]. Should the results of Numabe et al. [154] be also a potent stimulant of sperm motility [165,166]. The confirmed, this could be a promising approach to molecular mechanism of motility stimulation, however, improve fertilization rates especially for endangered works via an A -receptor-mediated activation of adenylate 2 species. cyclase [167], which in turn is thought to enhance the intracellular cAMP concentration [168,169]. Because of The conflicting results on the effectiveness of a pentoxifyl- this characteristic, the use of 2-DA in assisted reproduc- line treatment raised the question of the embryotoxicity of tion programs has been discussed. Other studies, how- this substance, especially since possible pentoxifylline- ever, failed to detect a responsive cAMP activity and either induced adverse effects on spermatozoa [157] and mouse stimulatory or inhibitory G-proteins in spermatozoa embryo development [158,159] have been reported. In [170,171]. In addition, Rivkees [172] could not detect contrast, Lacham-Kaplan & Trounson [160] did not A a- and A b-receptor gene expression in the rat testis. 2 2 observe such negative effects on embryonic development This would imply that these stimulatory receptors are after insemination of the oocytes with spermatozoa incu- either not present or, at least, at very low levels. On the bated in 3 mM pentoxifylline. Finally, short-term incuba- other hand, the cAMP-inhibitory A -receptor has a capac- 1 tion of spermatozoa with subsequent washing of the male itative effect on human spermatozoa when stimulated germ cells did not produce such adverse effects in intrau- with an agonist [173]. While Imoedemhe et al. [174] in an terine insemination or ICSI (Henkel et al., unpublished) internally controlled prospective study showed signifi- [156]. An alternative approach to increase sperm motility cantly higher fertilization rates after sperm stimulation or the number of motile spermatozoa was to administer with 2-DA and hence suggested further evaluation of the the drug orally over period of 3 to 6 months [161]. In a drug in assisted reproduction programs, Tournaye et al. placebo-controlled study including 47 normozoospermic [150] recommends a careful evaluation and selection of men with idiopathic asthenozoospermia, Merino et al. the patients before the treatment with motility stimulants. [162] showed a significant increase in progressive motility in men who received 1,200 mg of pentoxifylline per day Recently, 2-DA and pentoxifylline have been suggested for over 6 months. Clinical data about fertilization and preg- sperm stimulation in an in vitro culture of testicular tissue nancy, however, are still not available. in order to obtain motile spermatozoa for ICSI [175]. However, it seems rather questionable whether a treat- Another important point that must not be underesti- ment of spermatozoa with 2-DA for the purpose to ferti- mated in explaining the controversial effects of pentoxifyl- lize oocytes should be recommended because there is also line is the fact that this drug is an unspecific inhibitor of evidence that it has adverse effects on embryos. In this the phosphodiesterase (PDE). Considering that eleven regard, 2-DA significantly reduced the cleavage of mouse different families of this enzyme have been described embryos beyond the 2-cell stage [160]. In addition, 2-DA [163], of which PDE-1 and PDE-4 are present in human and cAMP have been shown to exert cytotoxic effects by spermatozoa and stimulate different sperm functions, i.e. inducing G1 cell cycle arrest [176]. In view of this, a ther- acrosome reaction and motility, respectively [164], an apeutic clinical use of stimulants that increase the intrac- Page 10 of 22 (page number not for citation purposes)