1 Discipline of Biological Sciences and 2 ARC Centre of Excellence in Biotechnology and Development, University of Newcastle, NSW 2308, Australia
3 To whom correspondence should be addressed at: Discipline of Biological Sciences, School of Environmental and Life Sciences, Callaghan, NSW 2308, Australia. Email: jaitken{at}mail.newcastle.edu.au
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Abstract |
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Key words: DNA damage/electrophoretic method/human spermatozoa/morphology/sperm isolation
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Introduction |
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While techniques such as swim-up and density gradient centrifugation perform adequately with normal semen samples containing large subpopulations of vigorously motile spermatozoa, these techniques are time consuming and do not avoid the damaging effects of centrifugation. In light of these considerations, we have evaluated the feasibility of a novel approach to sperm isolation, based on the electrophoretic separation of these cells on the basis of their size and charge.
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Materials and methods |
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Semen samples
Human semen samples were obtained from 31 healthy donors of unknown fertility status after at least 48 h abstinence. Samples were allowed to liquefy at room temperature and were then subjected to a routine semen analysis using the guidelines set out by the World Health Organization (1999). Results of these analyses indicated that the majority of samples were in the high normospermic range (sperm concentration 52 ± 5.2 x 106; vitality 83 ± 1.5%; motility 72 ± 2.1%). All procedures were approved by the University of Newcastle Human Ethics Committee.
Electrophoretic sperm isolation
The electrophoresis-based Microflow® technology for the separation of spermatozoa by size and charge consists of four separate compartments: two inner chambers (inoculation and collection) and two outer chambers. The outer chambers were separated from the inner chambers by two polyacrylamide restriction membranes with a pore size of 15 kDa (Life Therapeutics, Sydney, Australia), which allowed the movement of small molecules, water and ions between the inner and outer chambers and yet retained the cell suspension within the inner chamber. The outer chambers housed the platinum-coated titanium mesh electrodes and two 12 V buffer pumps (one for each electrode chamber) running at 5 V, to circulate buffer through the chambers at a flow rate of 1.6 l/min. The two inner compartments comprised a 400 µl inoculation chamber into which semen is deposited and a 400 µl collection chamber containing buffer. These two chambers were separated by a polycarbonate separation membrane (5 µmol/l) with an active membrane area of 30 x 15 mm (Figure 1A). The pore size was chosen to permit the transit of spermatozoa across the polycarbonate membrane while excluding larger cells (leukocytes and/or precursor germ cells) that commonly contaminate the human ejaculate.
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Percoll gradient centrifugation and repeated BiggersWhittenWhittingham centrifugation
Percoll® fractionation was performed on a discontinuous two-step Percoll gradient. For this procedure, an isotonic solution was prepared by adding 90 ml of Percoll (Pharmacia LKB, Uppsala, Sweden) to 10 ml of 10 x Ham's F10 (ICN Biochemicals) supplemented with 100 mg of polyvinyl alcohol (PVA), 3 mg of sodium pyruvate, 0.37 ml of a 60% sodium lactate syrup and 200 mg of sodium hydrogen carbonate to give an isotonic preparation that was designated 100% Percoll (Lessley and Garner, 1983). This solution was diluted 1:1 with HEPES-buffered (20 mmol/l) BiggersWhittenWhittingham (BWW) medium (Biggers et al., 1971
), supplemented with 1 mg/ml PVA and discontinuous gradients were created by layering 3 ml of this low-density Percoll preparation above 3 ml of isotonic Percoll. Liquefied semen was layered onto the gradient and centrifuged for 20 min at 500g. Spermatozoa were then recovered from the base of the gradient and, for the chemiluminescence experiments, the low-density/high-density Percoll interface. Sperm counts were then performed and cells resuspended to a final concentration of 5 x 106/ml for further analysis. Similarly, for the repeated centrifugation protocol, samples were centrifuged for 5 min at 500g, following dilution of the semen 1:1 with HEPES-buffered (20 mmol/l) BWW medium supplemented with 1 mg/ml PVA. The pellet was subsequently resuspended in 6 ml of BWW and the centrifugationresuspension cycle repeated a further two times. Sperm counts were finally performed and, unless otherwise indicated, the spermatozoa resuspended to a final concentration of 5 x 106/ml for further analysis.
Sperm and round cell number
Cell counts were conducted using an approved Neubauer haemocytometer following dilution with a formalin/bicarbonate diluent, as specified by the World Health Organization (1999).
Vitality
The vitality of sperm suspensions was assessed using the eosin dye exclusion test. Ten millilitres of 0.05% eosin dye solution in phosphate-buffered saline (PBS) was mixed with 10 ml of sample and examined by phase-contrast microscopy at 400x magnification. A minimum of 200 cells was scored for each sample and results recorded as percentage of live cells.
Sperm motility
Spermatozoa were wet-mounted on slides pre-warmed at 37°C and assessed for the percentage of motile cells using phase-contrast microscopy. For this purpose, at least 200 cells were scored immediately after preparation at 400x magnification.
Computer-assisted sperm assessment
A 10 ml sample of spermatozoa was aliquoted onto a pre-warmed disposable slide with a fixed chamber depth 30 mm. Motion parameters were then captured using a 240 V B/W CCD Camera (Panasonic, Belrose, NSW, Australia) at a frame rate of 50 Hz and recorded using dark-field illumination on professional-grade Super VHS videotape with a Super VHS videotape recorder (JVC, Yokohama, Japan). Samples were evaluated using the HTM-IVOS (Hamilton-Thorn Corporation, Danvers, MA, USA) computer-aided sperm analyser (CASA). The parameter settings applied were as follows: frames = 30 at 50 Hz; minimum contrast = 10; minimum size = 3; non-motile head size = 5; non-motile intensity = 90; threshold average path velocity value = 25 µm/s; slow cells motile = no. A minimum of 100 cells was analysed per treatment population.
Morphology
Samples were smeared onto pre-prepared poly-L-lysine coated slides and allowed to dry at room temperature. Dried smears were fixed in 95% ethanol (Fronine, Riverstone, NSW, Australia) for 15 min then stained by a modification of the Papanicolaou method, as described by the World Health Organization (1999). Smears were re-hydrated for 3 min in 50% ethanol, rinsed for 10 s in dH2O and stained with Harris' haematoxylin (Fronine) for 3 min. Smears were then washed twice with running tap H2O for 5 min, separated by an acid ethanol (0.25% HCl in 70% ethanol) treatment for 2 s. Following a brief 1 s dip in dH2O, smears were progressively dehydrated in 50, 70, 80 and 95% ethanol for 10 s then incubated with Orange G6 (Fluka, Buchs SG, Switzerland) cytoplasmic stain for 2 min. Surplus stain was removed by 95% ethanol for 20 s and EA-50 cytoplasmic and nucleolar staining performed for 5 min. Smears were then dehydrated (95% ethanol for 15 s followed by 100% ethanol for 2 min), allowed to completely air-dry and mounted using DPX media (BDH laboratory supplies, Poole, UK). Morphological examination was performed using a Zeiss Axioplan 2 (Ziess, Oberkochen, Germany) microscope using a 100x oil-immersion objective and a total magnification of 1250x.
The classification and evaluation of sperm morphology was established according to Menkveld et al. (1990). The entire spermatozoon (head, neck, midpiece and tail) was taken into consideration for evaluation along with any germinal epithelium or other cell types present. Spermatozoa were classified into one of seven groups, normal (whole sperm), large, small, elongated, duplicated and amorphous heads, all with or without the presence of a cytoplasmic droplet and/or tail, neck and/or midpiece defect (Menkveld et al., 1990
). The seventh group consisted of spermatozoa with a normal head with a tail and/or a neck and/or a midpiece defect and/or the presence of a cytoplasmic droplet (Menkveld et al., 1990
). Tail, neck and midpiece defects, loose heads, germinal epithelium and unknown cells were recorded separately and expressed per 100 spermatozoa (Menkveld et al., 1990
). In addition, the morphological normality of the sperm preparations was assessed using the sperm deformity index (SDI) as described by Panidis et al. (1998)
. For each analysis, a minimum of 100 spermatozoa was analysed with the aid of an eyepiece micrometer.
Chemiluminescence assessment of leukocyte contamination
Semen samples were subjected to a 5 min electrophoretic separation as described previously. At the end of the separation period the separated sperm suspension and the residual semen samples were washed twice in BWW (500g for 5 min) prior to chemiluminescence analysis. This wash step was necessary to remove any traces of the antioxidants present in seminal plasma that would have artificially suppressed the chemiluminescent signals generated by the residual sperm population. In order to control for the centrifugation process itself, the separated sperm populations were also centrifuged under identical conditions so that the signals generated by the separated and residual sperm populations could be directly compared. Luminol-dependent chemiluminescence was recorded on a Berthold 953 luminometer (Berthold Detection Systems GmbH, Crown Scientific Pty Ltd, Moorebank, Australia) at 37°C using 400 µl aliquots of spermatozoa at a concentration of 2 x 106/ml. Cell-free medium and dimethyl sulphoxide (DMSO) vehicle controls were recorded with every treatment replicate to ensure sperm suspension-dependent responses. Luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione) (10 mmol/l) was prepared in DMSO and diluted with sperm suspensions to give a final concentration of 100 µmol/l. Luminol was supplemented with horseradish peroxidase, freshly prepared as a 2 mg/ml stock solution in electrophoresis buffer, 8 µl of which was added to 400 µl of sperm suspension to give a final peroxidase activity of 11.52 U/ml. Any contaminating leukocytes were activated after 5 min incubation by addition of 20 µl zymosan opsonized with autologous serum and results were recorded as continuous traces and as integrated photon counts over a fixed time period of 20 min. This method gives a linear relationship between chemiluminescence and the concentration of CD45 positive leukocytes over the concentration range (103106 leukocytes/ml) typically encountered in human semen samples (see Figure 3C).
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Statistical analysis
Each experiment was repeated at least three times on independent samples and statistical analysis was performed using Microsoft Excel® 2000 and SuperANOVA (Abacus Concepts Inc., Berkeley, CA, USA). Angular transformations were carried out for percentages prior to statistical analysis using an angular transformation table, where p = sin2. Averages were calculated for each experiment, as well as standard errors of the mean (SEM) for n-1. Post-hoc testing was performed using Fisher's Protected Least Significant Difference (PLSD) and samples with a P-value of <0.05 were considered significant.
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Results |
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Morphology
An analysis of sperm morphology indicated that the spermatozoa separated using the electrophoretic system possessed a significantly improved morphology compared with the residual excluded population (Figure 4A; P<0.001). This difference was consistent across all time intervals assessed and did not vary significantly with the duration of the electrophoretic treatment. These data were reinforced using an alternative technique for assessing morphology, the sperm deformity index (SDI) introduced by Panidis et al. (1998). This assessment technique has been shown to provide a correlation between the morphological status of a given sperm population and its potential for fertilization in vitro. In this study, the SDI values recorded for the separated sperm populations was significantly below the threshold SDI value of 0.93 (Panidis et al., 1998
) for all electrophoretic time-points (Figure 4B; P<0.001). As SDI values increase above this threshold, the fertilizing capacity of the spermatozoa is held to decrease proportionally (Panidis et al., 1998
). The ability of the electrophoretic technique to select a subpopulation of morphologically normal spermatozoa with low SDI values therefore indicates enrichment for spermatozoa with potentially enhanced fertilizing potential.
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In terms of sperm recoveries, no significant difference was observed between the average number of spermatozoa isolated by electrophoresis (11.42 ± 1.59 x 106/ml) and Percoll gradient centrifugation (11.92 ± 1.42 x 106/ml). Significantly higher yields were observed using the 3xcentrifugation method (Figure 8A; P<0.001), as might be expected given the non-selective nature of this sperm preparation technique. However, these yields were accompanied by the presence of contaminating round cells (2.67±0.60x106/ml) not observed with the aforementioned techniques.
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TUNEL analysis revealed that the spermatozoa prepared by electrophoresis and Percoll gradient centrifugation exhibited less DNA damage than those prepared by repeated centrifugation. However, only in the case of the electrophoretically isolated spermatozoa was the level of DNA damage observed significantly lower than that observed in the original ejaculate (P<0.05).
Analysis of the morphological normality of the spermatozoa by ANOVA revealed a significant difference due to treatment; the electrophoretically separated cells exhibiting significantly more normal forms than the other preparative techniques analysed (Figure 9A; P<0.05). Furthermore, analysis of morphology using the sperm deformity index showed a significant reduction in sperm abnormalities for cells separated using the electrophoretic system compared with alternative preparation procedures as well as the original ejaculate (Figure 9B; P<0.05). Particular attention was placed on small-headed spermatozoa that, because of their shape, might have passed across the 5-µm filter more readily than normal cells, despite their lack of morphological normality. However, this analysis revealed no significant difference in the incidence of such cells between electrophoretically separated sperm (14±1.5%), 100% Percoll-separated sperm (15±3.2%) and the original ejaculate (14±2%).
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Discussion |
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Our data indicate that high numbers of viable, motile spermatozoa exhibiting low levels of DNA damage and high levels of morphological normality can be isolated from human semen utilizing a sperm separation strategy based on sperm size and electronegative charge. The importance of cell size as a selection criterion is self-evident, spermatozoa being one of the smallest cells in the body and, importantly, significantly smaller that the major contaminants in human semen, precursor germ cell and leukocytes.
Although a low-level chemiluminescence signal was generated by the electrophoretically separated cells (Figure 3B), this could be attributed to the washing steps that had to be employed prior to chemiluminescence in order to permit direct comparison of the residual and separated sperm populations. The physical shearing forces associated with repeated centrifugation have previously been shown to enhance the chemiluminescent activity of human spermatozoa (Aitken and Clarkson, 1988). In the absence of centrifugation, the electrophoretically separated sperm populations did not generate a chemiluminescence signal in response to zymosan (Figure 3B). Such results emphasize how minimally traumatized these electrophoretically prepared sperm suspensions were (Figure 3B) and the extent to which leukocyte contamination had been avoided.
The importance of cell charge is in keeping with existing data indicating that normal human spermatozoa possess a glycocalyx that is rich in sialic acid residues (Kallajoki et al., 1986; Caldaza et al., 1994
). The existence of a positive correlation between the electro-negativity of human spermatozoa and the quality of these cells may be partly related to the presence of CD52, a highly sialated, lipid-anchored molecule that is acquired by spermatozoa during epididymal transit (Kirchhoff and Schroter, 2001
). Thus, the negative charge associated with high quality spermatozoa may simply reflect the fact that these cells have differentiated normally in the testes and entered the epididymis in a sufficiently operational state to participate in the massive cellcell transfer of GPI-anchored CD52 to the sperm surface (Schroter et al., 1999
). This molecule is thought to play an important role in the capacitation of human spermatozoa. As sperm capacitate, CD52 moves from a widely distributed surface pattern to become concentrated in the equatorial region of these cells. Male infertility is associated with a significant decrease in the percentage of spermatozoa expressing CD52 and a reduced percentage of cells exhibiting the equatorial pattern of localization following capacitation (Giuliani et al., 2004
). Furthermore, this pattern of CD52 expression was found to be highly correlated with sperm morphology (Giuliani et al., 2004
), suggesting at least one reason why electrophoretically separated spermatozoa might be characterized by excellent morphology. Preliminary experiments have established that neuraminidase treatment of human spermatozoa interferes with their isolation using the electrophoretic system (C.Ainsworth, unpublished observations). Such results are in keeping with the proposed importance of sialation in establishing the charge differences that underpin the electrophoretic method of sperm separation. The involvement of CD52 in establishing the overall sialation status of human spermatozoa is currently under investigation.
Whatever biochemical principles underpin the electrophoretic sperm isolation procedure, this method is clearly capable of isolating large numbers of spermatozoa that are viable, motile, morphologically normal and relatively free of DNA damage and contaminating cells. Electrophoretic separation compared favourably with Percoll gradient centrifugation in terms of the purity of the sperm population, the lack of ROS generation, as well as the vitality and morphological normality of the isolated cells. The electrophoretic procedure was also superior to Percoll gradient centrifugation in terms of the time taken to isolate the spermatozoa and the lack of physical trauma. The fact that neither a centrifuge nor a skilled technician are necessary for this sperm isolation procedure also means that it could be readily adopted as an office procedure in order to isolate spermatozoa for assisted conception purposes. In principle, a semen sample would simply have to be inoculated into one chamber of the cassette, current applied for 5 min and the isolated spermatozoa removed, diluted with culture medium and used for therapeutic purposes.
The lack of DNA damage is a particularly valuable feature of the electrophoretic sperm separation technique. DNA damage in the male germline has been associated with impaired fertility following natural conception; the time to pregnancy increasing as a function of the proportion of sperm with abnormal chromatin (Spano et al., 2000; Loft et al., 2003
). Furthermore, DNA damage in spermatozoa has been linked with poor conception rates following IVF (Sun et al., 1997
; Aitken, 2004
). However, this is not consistently the case with ICSI (Lewis and Aitken, 2004
). With this technique, successful fertilization can be achieved despite high levels of DNA damage in the injected spermatozoon, regardless of whether ejaculated or testicular spermatozoa are used in the course of therapy (Twigg et al., 1998a
; Aitken, 2004
; Gandini et al., 2004
; Lewis and Aitken, 2004
). This might be expected, since with ICSI fertilization does not depend on the functional competence of the spermatozoa in terms of the ability of these cells to capacitate, acrosome react, penetrate the zona pellucida and fuse with the vitelline membrane of the oocyte. However, while ICSI might permit fertilization with DNA-damaged spermatozoa, such success might be achieved at some cost to the embryo. According to recent studies, the post-fertilization development of human embryos can be seriously impaired by the use DNA-damaged spermatozoa in assisted conception (Bungum et al., 2004
; Lewis and Aitken, 2004
; Virro et al., 2004
). Such disruption of embryonic development is presumably associated with the abortive transcription of damaged genes originating from the paternal genome, as well as possible epigenetic effects (Braude et al., 1988
; Tesarik et al., 2001
). Thus, the ability of the electrophoretic sperm isolation method to select germ cells exhibiting low levels of DNA damage with great rapidity and efficacy should be particularly valuable in the delivery of assisted conception services.
Notwithstanding the ability of the electrophoretic system to isolate spermatozoa enriched for normal morphology and low levels of DNA damage, this procedure did not enrich for sperm motility. This is not an unexpected finding, because previous studies have found that the electrophoresis of spermatozoa is detrimental to their motility (Engelmann et al., 1988). In the comparative wing of this study, the quality of sperm motility, as assessed by CASA, was no different regardless of whether the spermatozoa were prepared by Percoll gradient centrifugation or the electrophoretic method. However, there was a decrease in the percentage of motile spermatozoa with the latter, compared with the Percoll technique. We interpret these findings as indicating that electrophoresis of spermatozoa disrupts motility in a subset of vulnerable spermatozoa, possibly by interfering with the regulation of ion fluxes across the sperm plasma membrane. If only moribund spermatozoa are negatively affected by the electrophoretic procedure then there should be little impact on the fertilizing capacity of the sperm suspension as a whole. Clinical trials are being conducted to address this issue in the context of assisted conception therapy. Of course, such factors are of little concern if ICSI is to be performed. Indeed, one of the exciting prospects for this method of sperm isolation, is its potential application in the isolation of spermatozoa exhibiting low levels of DNA damage from complex cellular mixtures such as testicular or epididymal biopsies. In principle this electrophoretic sperm isolation procedure has great potential as an extremely versatile, time- and cost-effective method for preparing spermatozoa for a wide variety of assisted conception applications. It must be emphasized, however, that this proof-of-principle study has been confined to the analysis of normospermic donors. Additional studies will be required with seriously compromised, pathological semen samples to confirm the effectiveness of this electrophoretic technique in the management of male infertility.
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Acknowledgements |
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References |
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Submitted on March 9, 2005; accepted on March 10, 2005.