Hum. Reprod. Advance Access originally published online on March 2, 2007
Human Reproduction 2007 22(5):1405-1412; doi:10.1093/humrep/dem015
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Molecular glass wool filtration as a new tool for sperm preparation
1 Department of Dermatology/Andrology Unit, University of Leipzig, Leipzig, Germany 2 Department of Dermatology and Andrology, Justus Liebig University, Giessen, Germany
3 To whom correspondence should be addressed at: Department of Dermatology, Training Center of the European Academy of Andrology, University of Leipzig, Philipp-Rosenthal-Strasse 23-25, 04103 Leipzig, Germany. Tel.: +49 341 9718611; Fax: +49 341 9718619; E-mail: uwe.paasch{at}medizin.uni-leipzig.de
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Abstract |
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BACKGROUND: Magnetic-activated cell sorting (MACS) using annexin V-conjugated microbeads in a liquid phase eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine (EPS) residues. The procedure allows the enrichment of a sperm population free of apoptosis markers, giving higher fertilization potential. Our aim was to determine if the annexin V binding principle can be transferred onto a glass wool filter system in order to produce a solid phase filter.
METHODS: Semen samples (n = 42) were subjected to a molecular glass wool filter system using glass surfaces coated with annexin V and compared with aliquots separated by conventional glass wool, as well as with annexin V-MACS. The extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labelled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and EPS using a fluorescein isothiocyanate-coupled monoclonal antibody.
RESULTS: Annexin V-negative sperm filtered out by the newly developed molecular glass wool filtration (GWF) system displayed superior quality in terms of high MMP integrity, as well as, to a small extent, caspase 3 activation and EPS.
CONCLUSIONS: The effect of traditional GWF can be further improved by combination with annexin V binding. This newly developed solid phase molecular filter system has been proven to enrich spermatozoa free of apoptosis markers to the same extent as the annexin V magnetic separation technique. The selection of spermatozoa free of apoptosis markers by molecular glass wool filters may enhance the results of IVF.
Key words: annexin V/apoptosis marker/glass wool/IVF/sperm
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Introduction |
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The application of assisted reproduction techniques (ARTs) has provided help to many men seeking to father a child, although the current success rates of these procedures remain suboptimal (Society for Assisted Reproductive Technology and American Society for Reproductive Medicine, 2004
). Successful assisted reproduction in male infertility is mainly dependent on the quality of the sperm sample (Ombelet et al., 2003). Although the presence of apoptosis in ejaculated sperm when compared with somatic cells still remains unconfirmed (Sakkas et al., 1999, 2002), ejaculate from infertility patients are of low quality, as measured by standard semen parameters and apoptosis-like characteristics, e.g. activated caspases, externalization of phosphatidylserine (EPS) at the outer plasma membrane, DNA-fragmentation and disruption of the mitochondrial membrane potential (MMP) (Comhaire et al., 1988; Glander and Schaller, 1999; Oehninger et al., 2003; Paasch et al., 2003b; Grunewald et al., 2005).
A variety of sperm preparation techniques have been proven to select spermatozoa that are characterized by superior motility and morphology and are capable of fertilizing the oocyte. Among them, the double density gradient centrifugation (DGC) and the swim-up procedure are currently used as standard preparation techniques. In addition, the glass wool filtration (GWF) technique is known to provide sperm samples with comparable recovery rates, motility, morphology and fertilizing capacity if appropriate types of glass wools used (Holmgren and Jeyendran, 1993; Johnson et al., 1996; Van Den et al., 1997; Henkel and Schill, 2003;). The one-step washing technique is considered an alternative for processing certain compromised samples (Srisombut et al., 1998). Recent studies revealed that samples processed by DGC and GWF showed significantly decreased levels of caspase activation, disrupted MMP, DNA-fragmentation and EPS when compared with unprocessed semen (Larson et al., 1999; Ollero et al., 2001; Paasch et al., 2003a; Rasch et al., 2005; Barroso et al., 2006).
The main apoptotic event detectable at the sperm surface is the externalization of the phospholipid PS, which is usually present only on the inner leaflet of the sperm plasma membrane (Vermes et al., 1995; Oosterhuis and Vermes, 2004). Annexin V is a phospholipid-binding protein that has high affinity for PS and lacks the ability to pass through an intact sperm membrane (van Heerde et al., 1995). Therefore, annexin V binding to spermatozoa may be used to label sperm that have compromised membrane integrity and that are less capable of fertilizing oocytes (Glander and Schaller, 1999).
Annexin V-conjugated super-paramagnetic microbeads can effectively separate spermatozoa free of apoptotic markers from those with deteriorated plasma membranes based on the EPS using magnetic-activated cell sorting (MACS). MACS separation of sperm yields two fractions: annexin V-negative (intact membranes, spermatozoa free of apoptotic markers) and annexin V-positive (EPS, spermatozoa-labelled positive for apoptotic markers) which is retained in the magnetic field (Grunewald et al., 2001; Paasch et al., 2003b). Sperm preparation that combines MACS with double density centrifugation provides spermatozoa of higher quality, in terms of motility, viability and apoptosis indices, compared with other conventional sperm preparation methods (Said et al., 2005b). Furthermore, sperm prepared according to this protocol showed an improved ability to fertilize oocytes using the hamster oocyte penetration assay (Said et al., 2005c).
Although there is a major potential advantage in integrating annexin V-MACS in standard semen preparation protocols, at this time, the possibility of an accidental transmission of super-paramagnetic microbeads into oocytes can not be fully excluded. Therefore, a sperm preparation system using the binding properties of annexin V-without free flotation of super-paramagnetic microbeads in a liquid phase buffer is needed. The development of an annexin V-based solid phase separation system might be the next step towards selection of vital spermatozoa with superior fertilizing capacity.
In this study, we present a newly developed solid phase molecular filtration system which combines classical GWF with PS-binding properties of annexin V. The separation effect of the system was evaluated in terms of apoptosis-related parameters. Semen samples prepared by simple wash, standard GWF and annexin V-MACS served as controls.
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Experimental design
This study was approved by the Institution Review Boards of the Faculty of Medicine, University of Leipzig. Semen samples were obtained from 42 healthy donors of unproven fertility following a period of 3–5 days of sexual abstinence. Semen analysis was performed according to the World Health Organization (WHO) guidelines (World Health Organization, 1999
). Samples with 
50 x 106 spermatozoa-ml–1 and at least 50% progressive sperm motility were selected for the study. After liquefaction, the semen samples were washed by centrifugation and re-suspended in 1 ml human tubal fluid (HTF) media. The lowest number which can be processed has not been tested yet. One aliquot of the sperm suspension served as a control each time (simple HTF wash only). When the total sperm count exceeded 100 x 106 spermatozoa (n = 14) one extra aliquot was subjected to cryopreservation. The sperm suspensions (neat as well as cryopreserved) were further subdivided into four aliquots for investigation of the various sperm preparation techniques: (i) conventional GWF using raw glass wool, (ii) GWF using the commercially available SpermFertil® columns (TransMIT GmbH, Giessen, Germany), (iii) annexin V GWF and (iv) annexin V-MACS separation.
Activated caspase 3 levels, integrity of the MMP and extent of EPS were assessed in the control aliquot (simple HTF wash) and in all aliquots following the different preparation procedures (Figure 1).
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Cryopreservation—thawing protocol
The semen samples were cryopreserved with freezing medium TEST yolk buffer, consisting of TES (N-tris[hydroxymethyl]-methyl-2-aminoethanesufonic acid), Tris (Tris[hydroxymethyl]aninomethane) and chicken oocyte yolk (TYB, 20% oocyte yolk and 12% glycerol, Irvine Scientific, Santa Ana, CA, USA, Catalogue No 9971), conditions known to result in the best vitality parameters (motility, morphology and membrane integrity) of spermatozoa after cryopreservation, as demonstrated previously (Glander and Schaller, 1999
; Hallak et al., 2000). For cryopreservation, the semen samples were diluted drop-wise with an equivalent volume of TYB containing 12% (v/v) glycerol. The samples were placed into 2.0 ml cryotubes (Faust Laborbedarf AG, Schaffhausen, Switzerland) and frozen with the Nicool LM 10 system (Air Liquide, Wiesbaden, Germany). A slow cooling at level 2, which decreased the temperature from +24°C to +5°C within 15 min, was followed by incubation for 15 min at level 10, decreasing the temperature to –70°C (McLaughlin et al., 1990). Finally, the tubes were plunged into a liquid nitrogen tank (Biosafe MD, Cryotherm Kirchen, Germany) and stored at –196°C in the gas phase. Vials were thawed by incubating at 37°C for 20 min. Spermatozoa were washed and re-suspended in HTF media immediately after thawing, and sperm motility was re-assessed.
Isolation of spermatozoa with raw glass wool and the GWF SpermFertil® columns
SpermFertil® GWF columns were purchased from TransMIT GmbH. Before use, the columns were rinsed once with 5 ml HTF media. Following this, the 2 ml of the semen sample was loaded on top of the column.
Manufacturing of annexin V GWF columns
SpermFertil® columns (TransMIT GmbH) were first stored for 48 h at 40°C in a damp chamber and secondly incubated with 300 µl of poly-L-lysine (1 mg ml–1) for 72 h at room temperature. After washing with phosphate-buffered saline (PBS, pH 7.4), the glass wool was incubated for 1 h at room temperature with 200 µl 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP) (1 mg ml–1 in PBS). Reduction of the dithiogroup was achieved by the addition of 500 µl dithiothreitol (DTT) (50 mM in PBS) for 1 h at room temperature. After thorough washing with PBS, incubation with 200 µl of SPDP (1 mg ml) in sodium acetate buffer (SAB) (50 mM, pH 4.5) for 1 h at room temperature followed. Finally, washing with SAB, incubation with annexin V (100 µg ml in PBS) and subsequent washing with PBS plus 1 mM DTT completed the synthesis of annexin V glass wool columns.
The successful fixation of annexin V to the glass wool was evaluated by the relative presence of annexin V on the glass wool surface of an untreated SpermFertil® column in comparison to an annexin V glass wool column. Saturation of annexin V binding was achieved at 100 µg ml–1. Both columns were incubated with 1 mL HTF media containing 1% bovine serum albumin for 1 h at room temperature to avoid non-specific bindings. After being washed with 1 ml HTF media, the columns were incubated with 1 mL fluorescein isothiocyanate (FITC)-labelled anti-human annexin V and clone VAC-
diluted 1:20 in PBS pH 7.4 (Bender MedSystems, Vienna, Austria) for 20 min at room temperature. Subsequently, both columns were rinsed with 5 ml HTF media. Glass wool aliquots from both columns were prepared on glass slides, covered with a cover slip and evaluated by fluorescence microscopy (Figure 2A and B).
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Isolation of spermatozoa with deteriorated membranes by annexin V-MACS
Spermatozoa were incubated with annexin V-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at room temperature. About 100 µl of microbeads were used for each set of 10 x 106 separated cells. The sperm/microbeads suspension was loaded in a separation column containing iron globes, which was fitted in a magnet (OctoMACS; Miltenyi Biotec). The fraction composed of apoptotic spermatozoa was retained in the separation column and labelled as annexin V-positive, whereas the fraction with the intact membranes that was eluted through the column was labelled as annexin V-negative. The power of the magnetic field was measured as 0.5 T between the poles of the magnet and up to 1.5 T within the iron globes of the column. After the column was removed from the magnetic field, the retained fraction was eluted using annexin V-binding buffer (Miltenyi Biotec).
Detection of activated caspase 3
Levels of activated caspase 3 were detected in spermatozoa using a fluorescein-labelled inhibitor of caspase (FLICA) which is cell permeable, non-cytotoxic and binds covalently to active caspase 3 (Ekert et al., 1999; Bedner et al., 2000). The inhibitor was used with the appropriate controls according to the kit instructions provided by the manufacturer (Carboxyfluorescein FLICA, Immunochemistry Technologies, Bloomington, MN, USA). A 150-fold stock solution of the inhibitor was prepared in dimethylsulphoxide and further diluted in PBS to yield a 30-fold working solution. All test aliquots and controls (with 100 µl PBS) were incubated at 37°C for 1 h with 10 µl of the working solution and subsequently washed twice with the rinse buffer.
Evaluation of MMP
A lipophilic cationic dye (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine chloride) was used to determine the MMP in spermatozoa (ApoAlert Mitosensor KitTM, Clontech, CA). Spermatozoa with intact mitochondria (preserved MMP) emit an intense red fluorescence due to the formation of the dye aggregates. Dye aggregates cannot be formed in the absence of the membrane potential. The lipophilic cationic dye remains in its monomeric state and emits a green light marking the presence of sperm with a disrupted mitochondrial membrane. The kit was used according to the instructions of the manufacturer. Briefly, all aliquots were incubated at 37°C for 20 min in 1 µg of the lipophilic cation diluted in 1 ml PBS. Negative controls were processed identically for each fraction except that the stain was replaced with 10 µl PBS. Positive controls were performed using betulinic acid (60 µg ml–1) (Paasch et al., 2004b).
Measurement of PS externalization
EPS was examined using a monoclonal mouse anti-human PS antibody, clone 1H6 (Upstate Cell Signaling Solutions, Lake Placid, NY, USA). Spermatozoa were incubated with the PS antibody at a final concentration of 0.5 µg ml–1 in PBS containing 2% bovine serum albumin (PBSB) for 20 min on ice, followed by addition of 150 µl PBSB and centrifugation at 300 g for 5 min at 20°C. After the supernatant was discarded, each sperm pellet was incubated with 50 µl of secondary antibody (goat anti-mouse immunoglobulin G, fluorescein conjugate, Upstate Cell Signaling Solutions, Lake Placid, NY, USA) on ice for 20 min and protected from direct light. A second washing step in PBSB (300 g for 5 min at 20°C) was performed to remove excess antibody that was not bound to the spermatozoal surface. For assessment by fluorescence-activated cell sorter (FACS), sperm pellets were diluted in 400 µl PBSB. Negative controls were processed identically for each fraction except that the antibody was replaced with equal amounts of PBS. Positive controls were performed by induction of membrane damage with liquid nitrogen. Control samples were briefly plunged into liquid nitrogen and immediately thawed.
FACS analysis
The extent of activated caspase 3, intact MMP and EPS were evaluated by FACS analyses. All fluorescence signals of the labelled spermatozoa were analysed by the FACScan (Becton Dickinson, San Jose, CA, USA). A minimum of 10 000 spermatozoa were examined for each assay at a flow rate of <100 cells s–1. The sperm population was gated using 90° and forward-angle light scatter to exclude debris and aggregates. The excitation wavelength was 488 nm, supplied by an argon laser at 15 mW. Green fluorescence (480–530 nm) was measured in the FL-1 channel and red fluorescence (580–630 nm) in the FL-2 channel. The percentage of positive cells and the mean fluorescence was calculated on a 1023-channel scale using the FACS software Expo32 ADC (Coulter, Krefeld, Germany).
Statistical analysis
The Wilcoxon test and the Mann–Whitney U-test were used to calculate the difference between samples. Hypothesis testing was two-tailed, and P values <0.05 were considered statistically significant. All values are given as mean ± SE. All calculations were performed with Statistica 6.0 software (StatSoft; Tulsa, OK, USA).
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Semen samples' evaluation
The mean ejaculate volume was 2.8 ± 0.2 ml, and the sperm concentration, total sperm count and percentage motility (WHOa+b) in the raw ejaculate were 88.1 ± 15.1 x 106 ml–1, 249.4 ± 54.9 x 106 and 52.5 ± 2.0%, respectively. After cryopreservation, motility (WHOa+b) decreased to 36.1 ± 3.1% (P < 0.001).
Evaluation of the annexin V glass wool columns
The fixation of annexin V on the surface of the glass wool was proven by the receipt of strong fluorescence signals from annexin V glass fibers after incubation with the FITC-labelled anti-human annexin V antibody (Figure 2A, red arrows). Owing to the physical characteristics of glass wool, the fluorescence signals were transmitted along the fibers resulting in a homogeneous fluorescence on their surface (Figure 2A). As a negative control, standard SpermFertil® columns showed almost no fluorescence (Figure 2B).
Activated caspase 3, MMP and EPS
Semen samples from healthy donors contained varying proportions of spermatozoa with activated apoptosis signalling (Table I). The percentage of caspase 3 positive (CP3+) spermatozoa was negatively correlated with the percentage of spermatozoa with intact MMP (R = –0.56, P < 0.01, Spearman's rank correlation) and positively correlated with the percentage of spermatozoa with EPS (R = 0.31, P < 0.01). The disruption of the MMP and the EPS to the sperm surface showed also a significant positive correlation (R = 0.30, P < 0.01).
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Impact of conventional GWF on apoptosis signalling
Filtration using raw glass wool significantly reduced the number of EPS+ spermatozoa in comparison to the HTF wash control (–4.9% EPS+ sperm, P < 0.05, Table I). However, the method apparently induced significant caspase 3 activation (+13.2% CP3+ sperm, P < 0.05), as well as elevated degrees of disruption of the MMP (+13.6% sperm with disrupted MMP compared with HTF wash control, P > 0.05). In contrast, GWF using SpermFertil® columns significantly reduced the number of EPS+ spermatozoa (–8.8% EPS + sperm, P < 0.01) with control without alteration of the MMP and without activation of caspase 3 (P > 0.05). Compared with the raw glass wool, spermatozoa separated by commercially available SpermFertil® columns contained significantly lower amounts of active caspase 3 (–20.8% CP3+ sperm, P < 0.05). The depletion of EPS+ sperm and those showing disrupted MMP was slightly improved compared with the raw glass wool columns (P > 0.05).
Characterization of the annexin V-negative subpopulation derived by MACS and molecular GWF
Both the annexin V GWF and annexin V-MACS separation techniques reduced the levels of annexin V-negative spermatozoa with active caspase 3, compared with control (–14.9% and –10.2%, respectively, P < 0.05, Table I). In addition, both annexin V-negative fractions were characterized by significantly higher amounts of spermatozoa with intact MMP in comparison to the control (annexin V GWF: + 9.2% MMP-intact sperm, annexin V-MACS: + 9.9% MMP-intact sperm, P < 0.05), as well as when compared with the conventional GWF methods (P < 0.05). Although there was no difference between both annexin V-based preparation methods in terms of caspase 3 activation and integrity of MMP (P > 0.05), the annexin V glass wool filtrated subpopulation showed improved reduction of EPS+ spermatozoa, compared with annexin V-MACS (–3.4% EPS+ compared with the MACS system). However, annexin V glass wool was as effective as SpermFertil® columns in decreasing the EPS+ sperm when compared with control
Within the annexin V-positive fraction after MACS, spermatozoa characterized by significantly increased caspase 3 activation, disruption of MMP and EPS were enriched (P < 0.05, compared with sperm washed in HTF, filtrated by raw and SpermFertil® glass wool as well as with the annexin V-negative subpopulations).
The separation effect of conventional- and molecular-based sperm preparation following cryopreservation and thawing
Following freezing and thawing of HTF wash samples, the percentage of spermatozoa displaying activated apoptosis signalling increased significantly: + 23.2% sperm containing active caspase 3, + 30.0% sperm with disrupted MMP and + 4.7% EPS+ sperm were detected in the cryopreserved samples (P < 0.01 compared with HTF in Tables I, and II).
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Preparation of the cryopreserved and thawed semen samples by filtration through raw glass wool had no significant impact on the levels of caspase 3 activation, MMP disruption and PS externalization (P > 0.05). As observed in neat semen samples (Table I), the SpermFertil® columns did not negatively affect the spermatozoa. Moreover, application of this commercially available glass wool resulted in a significant decrease in CP3+ spermatozoa (–20.1%) and those with disrupted MMP (-17.4%) after the freezing and thawing process (P < 0.01 versus HTF control), whereas the reduction of EPS+ spermatozoa was not significant.
After cryopreservation and thawing, the annexin V-negative fraction of ejaculate separated by MACS was characterized by significantly lower levels of CP3+ spermatozoa (–23.9%, P < 0.01) and EPS+ spermatozoa (–9.3%, P < 0.05) when compared with the HTF washed aliquots, as well as higher numbers of MMP-intact spermatozoa (+15.1%, P < 0.05).
Filtration of the cryopreserved and thawed aliquots through molecular annexin V glass wool decreased significantly the percentage of spermatozoa showing activated caspase 3 (–23.6% versus the control aliquots, P < 0.01). The reduction was equivalent to the results achieved in the MACS separated annexin V-negative fraction. However, the depletion of spermatozoa with EPS (–6.9%, P = 0.06) and disrupted MMP (–7.9%, P > 0.05) did not reach statistical significance compared with cryopreserved and thawed control samples washed in HTF only.
The annexin V-positive fraction after MACS was not investigated, as the enrichment of spermatozoa with an activated apoptosis signalling after cryopreservation and thawing in that fraction has been proven in several previous studies (Grunewald et al., 2001; Paasch et al., 2004c; Grunewald et al., 2005).
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Discussion |
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The increase in ART applications with concurrent low success rates has directed interest to the development of novel approaches of sperm preparation, such as electrophoretic separation and magnetic cell separation, which have demonstrated encouraging results for the isolation of higher quality spermatozoa (Ainsworth et al., 2005
). Annexin V-conjugated microbeads specifically target spermatozoa with deteriorated membranes that display EPS as a manifestation of apoptosis signalling (Paasch et al., 2003c, 2004a,b). Therefore, unlike routine sperm preparation techniques, sperm preparation using annexin V coupled super-paramagnetic microbeads acts on the molecular level.
This new sperm preparation strategy enables enrichment of vital spermatozoa free of apoptotic markers, especially when cryopreservation needs to be performed (Grunewald et al., 2001; Paasch et al., 2004c). The system can be easily integrated into standard semen preparation techniques, providing higher fertilization rates (Said et al., 2005b,c). However, the safety of this method has not been fully elucidated. In principal, the risk of accidental transmission of super-paramagnetic beads into oocytes cannot be fully eliminated.
Among the sperm preparation techniques available for ART, GWF is also known to result in semen samples with superior recovery rates, motility and morphology (Van Den et al., 1997; Henkel and Schill, 2003). Recently, samples processed by GWF using the SpermFertil® system have been demonstrated to enrich sperm with low levels of caspase 3 activation and EPS (Rasch et al., 2005).
In this study, the principle of combining the classical GWF method with PS-binding properties of annexin V has been investigated in neat and cryopreserved–thawed semen samples by measuring apoptosis signal transduction markers.
Effect of GWF on sperm quality and apoptosis markers
The impact of two types of conventional GWF systems has been investigated. Raw glass wool had a clear negative impact on semen samples. Caspase 3 activation and the disruption of MMP exceeded the levels measured in controls. Only when applied to cryopreserved samples was there a minor filtration effect in terms of apoptosis markers. Therefore, raw glass wool may not be used for sperm preparation prior to ART (Henkel and Schill, 2003). In contrast, the application of the commercially available SpermFertil® glass wool was perfectly tolerated by sperm and led to a depletion of spermatozoa with membrane damage. In earlier investigations, glass wool was found to be useful for removal of leukocytes in cases of leukocytospermia. A reduction of peroxidase-positive cells of up to 90% can be achieved (Sanchez et al., 1996). The mechanism by which GWF removes leukocytes and deficient spermatozoa with structurally abnormal and physically damaged membranes is thought to be by adherence to the glass wool fibers. In addition, the conventional SpermFertil® glass wool is at least partially able to separate spermatozoa with cryopreservation and thawing induced membrane damage, as well as following the activation of caspases and the disruption of MMP.
Development of a solid phase molecular GWF system
As a first approach towards a solid phase annexin V-based molecular sperm filter, recombinant human annexin V was covalently bound to SpermFertil® glass wool to combine the advantage of enrichment of annexin V-negative spermatozoa with the known high quality of sperm preparation by conventional GWF. Annexin V GWF is as easy to perform as conventional GWF and requires significantly less time and processing steps, compared to the MACS procedure.
Impact of annexin V separation systems on apoptosis signalling
The annexin V-negative fractions separated by MACS as well as by the newly developed molecular GWF system displayed a lower percentage of caspase 3 activation as well as higher mitochondrial membrane integrity, relative to the classical sperm preparation techniques. Conversely, the annexin V-positive fraction demonstrated the highest expression levels of these apoptotic markers which may serve as an internal control measure for the assessment of separation efficacy. MACS using annexin V microbeads has been repeatedly shown to efficiently isolate non-apoptotic spermatozoa with a superior fertilization capacity (Paasch et al., 2004c; Said et al., 2005c).
The technique, transferred to a solid phase GWF system, appears to be adequate and even highly efficient, since the number of spermatozoa with EPS was lowest in the annexin V-negative fraction after molecular GWF. Moreover, standard GWF using the SpermFertil® system showed comparable low rates of EPS. Minimal EPS occurred in the annexin V-negative fraction, whereas a sizeable number of spermatozoa that stained negative for PS were found in the annexin V-positive fraction. This may be attributed to the fact that binding sites are already blocked by the annexin V microbeads. In addition, other enzymes, such as protein kinases, and phospholipids, such as phosphatidylethanolamine, may serve as binding sites for annexin V, despite a high affinity for PS (Stuart et al., 1998; Muratori et al., 2004). This clearly indicates that the sperm quality can still be improved by integrating annexin V binding as a part of the sperm preparation protocol, which is concordant to our earlier studies (Said et al., 2005a).
In conclusion, annexin V glass wool is easy to use in molecular-based separation method with a high capacity to exclude spermatozoa with activated apoptosis signalling from semen samples. As the annexin V-based MACS was proven to enhance sperm quality and oocyte penetration capacity (Said et al., 2005c), application of annexin V glass wool may improve the outcome of ARTS. Although our data support the highly efficient filtration capacity of a solid phase annexin V coated glass wool filter, further development is needed for optimization.
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The authors would like to thank from the Clinical Andrology Laboratory University of Leipzig: Christian Kriegel and Gabi Kersten for technical assistance and Heidrun Janus for secretarial help. Funds from the German Research Council (Deutsche Forschungsgemeinschaft, DFG GL 199/4) and junior research grant from the Faculty of Medicine, University of Leipzig (formel.1-54).
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Submitted on November 21, 2006; accepted on January 9, 2007.
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