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Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa
Reproductive BioMedicine Online (2011) 22, 37– 43
www.sciencedirect.com www.rbmonline.com
ARTICLE
Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa T Ebner
a,*
, O Shebl a, M Moser a, RB Mayer a, W Arzt b, G Tews
a Landes- Frauen- und Kinderklinik, IVF-Unit, Linz, Austria; Institute of Prenatal Genetics, Austria
b
a
Landes- Frauen- und Kinderklinik,
* Corresponding author. E-mail address: [email protected] (T Ebner). Thomas Ebner, PhD, graduated with honours from the Paris Lodron University of Salzburg, Austria in 1992. After completing his doctorate and post-doctoral theses, he became a university lecturer in Salzburg. He has published more than 80 papers as first and co-author. Research interests include non-invasive IVF selection processes, laser application, vitrification and culture media. He was certified as a senior clinical embryologist in 2008. Currently he is scientific director of the European School of ART in Linz.
Abstract Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired
preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0–42.1%). Density gradient did not significantly improve the quality of spermatozoa selected (14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and, with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report on a sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: density gradient centrifugation, sperm chromatin dispersion test, sperm motility, strand breaks, Zech-selector
Introduction A relatively high number of patients fail to achieve pregnancy despite the obvious absence of a male or female
factor of infertility. It is likely that many of these couples actually present with a genomic male factor, including meiotic alterations, aneuploidy or sperm DNA damage (Sakkas and Alvarez, 2010). In particular, sperm DNA fragmentation
1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2010.09.004
38 is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome (Borini et al., 2006; Carrell et al., 2003; Duran et al., 2002; Evenson et al., 1999; Seli et al., 2004; Velez de la Calle et al., 2008; Zini et al., 2008). Several tests have been established for the analysis of sperm DNA fragmentation. Amongst others, TdTmediated-dUTP nick-end labelling (TUNEL) (Gorczyca et al., 1993), comet assay (Enciso et al., 2009; Hughes et al., 1996) and sperm chromatin structure assay (SCSA) (Evenson et al., 2002) as well as the sperm chromatin dispersion (SCD) test (Fernandez et al., 2003) are the approaches most commonly used in IVF laboratories. These assays can be subdivided into two categories, those directly detecting DNA damage (e.g. TUNEL) and those measuring DNA fragmentation after a rather mild denaturation process (e.g. SCSA, SCD). Although direct proof of strand breaks would be appreciated, all of the above mentioned tests unveil certain limitations (Bungum et al., 2004; Neguescu et al., 1998; Sakkas and Alvarez, 2010). One important aspect with respect to sperm DNA fragmentation is the question whether breaks are singleor double-stranded since single-stranded defects are probably easier to repair as compared with double-stranded DNA breaks (Sakkas and Alvarez, 2010). In this respect it should be kept in mind that the processing of spermatozoa could also cause an apparent increase in DNA strand breaks (Dalzell et al., 2004; Donnelly et al., 2001; Gosa ´lvez et al., 2009; Twigg et al., 1998); thus, the sperm processing technique applied for removing DNA-damaged spermatozoa is of utmost importance (Sakkas and Alvarez, 2010). Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa (Ahmad et al., 2007; Jackson et al., 2010; Marchesi et al., 2010), but, to date, there is no reliable approach to completely filter out spermatozoa with strand breaks from an ejaculate. This study was started in order to test the efficiency of a rather new sperm processing technique (Zech-selector) with respect to the reduction of spermatozoa with DNA damage.
Materials and methods During the study period, 39 patients with known male subfertility who presented at the study centre’s andrology laboratory for a second analysis of their ejaculate were recruited. The mean age of the men was 37.7 ± 6.5 years. An abstinence time of 3–5 days was recommended. All ejaculates were processed and analysed strictly according to the World Health Organization (WHO) manual (1999). Half of men suffered from isolated teratozoospermia (51%). A smaller percentage had isolated astheno- (8%) or oligozoospermia (8%). However, the remaining 33% of patients showed a drop in more than one sperm parameter, including five cases of oligoasthenoteratozoospermia (OAT). The study was approved by the Institutional Review Board of the Landes- Frauen- und Kinderklinik, Linz, Austria, and the patient received verbal information about the nature of the study. Participation in the study allowed the patients to receive the DNA analysis free of charge.
T Ebner et al. After control of liquefaction, the ejaculate was processed immediately in order to avoid excessive contact between seminal plasma and spermatozoa which could have altered chromatin packaging, thus possibly interfering with DNA staining. It was planned to make three analyses of DNA fragmentation per patient. After sterile masturbation, a small volume (about 25 ll) of raw semen was kept in order to have a reference value (sample 1). The rest of the ejaculate was split into two unequal parts in order to treat them differently. The first volume (1–2 ml) was processed using routine density gradient centrifugation technique (sample 2). In detail, semen was placed on the top of two layers (40% and 80%) of GM501 Gradient (Gynemed, Lensahn, Germany). After layering the sample was centrifuged at 180g for 20 min. Subsequently, both layers containing silane-coated colloidal silica were carefully removed and the pellet resuspended in BM1 medium (NMS Bio-Medical, Praroman, Switzerland). In order to reduce additional manipulation of the spermatozoa, only one centrifugation step was performed at 180g for 10 min. Finally, the purified sperm sample was incubated at 37C for approximately half an hour, to allow for swim-up, and then strandbreak measurement was performed. In parallel, a patented (European patent number 1,432,787) sperm selecting chamber (Zech-selector, AssTIC Medizintechnik GmbH, Leutsch, Austria) made of glass or polyethylene was filled with 1–3 ml of ejaculate (sample 3). These chambers accumulate an adequate number of motile spermatozoa without exposure to centrifugation stress (Ebner et al., 2003). In principle, both devices consist of two concentric wells which are overlaid by a U-ring and a cover glass (Figures 1 and 2) and progressive motile spermatozoa migrate from the ejaculate in the outer well to concentrate in the medium-filled inner well by using a capillary bridge created by the overlaying U-ring. After 1 h, a 25 ll sperm sample was taken from the central chamber and referred to further analysis. Patients whose ejaculate had to be processed for more than 1 h (e.g. due to delayed liquefaction) were excluded from the study for the sake of homogeneity of the study group. Thus, it could be guaranteed that all three samples (neat semen, density gradient and sperm selecting chamber) were analysed within 1 h (including time for liquefaction), in other words prolonged contact with seminal plasma was avoided. If the volume of ejaculate was large (>5 ml), both types of chambers (glass and polyethylene) were used giving four values in these patients. It has to be clarified that the only limitation with the present sperm preparation technique is that patients diagnosed with OAT were processed slightly differently. Since sperm count and progressive motility were reduced in these ejaculates, the number of spermatozoa migrating to the medium-filled inner well was reduced. Therefore, careful removal of the inner volume (in order not to cause contamination) and subsequent concentration of the motile spermatozoa by single-step centrifugation (10 min at 180g) was performed. It should be kept in mind that filling of the chambers can be tricky. It is important that a minimum volume of 2 ml is used for the polyethylene chamber and at least 3 ml should
Accumulation of DNA-strandbreak-free spermatozoa
a
39
b
Figure 1 Schematic cross-section of the sperm selector indicating the ejaculate in the outer ring (dots) and medium in the centre well (wave lines). Cover glass is not shown. (a) Sperm selector consists of the actual chamber (glass or polyethylene) and a U-ring (top left). (b) After filling of the chamber, the U-ring is inserted and creates a capillary bridge allowing motile spermatozoa to swim to the centre well (theoretical path of migration indicated by arrow).
Figure 2 The Zech-selector. The outer diameter of the glass chamber is 6.3 cm. The diameter of the inner circle, considered for concentration of motile spermatozoa, is 2 cm.
be filled in the glass chamber. If volume was less than required, the difference was filled up with BM1 medium. This worked without problems in the glass chamber, but in 2/23 (8.7%) polyethylene chambers, no contact between the ejaculate and the medium in the centre well could be achieved. This happened despite any possible drop in the volume of the polyethylene centre well being carefully placed. In addition, it turned out to be important to minimize movement of the U-ring once placed in order to avoid contamination of the inner well with immotile spermatozoa and other debris (due to suction forces). Theoretically, contamination can also occur if, after processing a certain volume of sperm suspension, is removed from the centre well for later analysis. In contrast to the polyethylene chambers (3/23 with contamination), the glass chambers (due to their construction) allowed for removal of the ejaculate from the outer ring, thus disconnecting the liquid bridge and avoiding suction forces. However, after a certain learning curve, filling of both chambers worked perfectly.
An improved SCD test, the so-called Halosperm assay (Halotech DNA SL, Tres Cantos, Spain), was used for the determination of the actual percentage of DNA-damaged spermatozoa. At the beginning of the study, processed sperm samples were also analysed with TUNEL assay. Since no difference in the percentage of DNA-fragmented spermatozoa was observed (Chohan et al., 2006), this additional evaluation was cancelled for financial reasons. Although the SCD test has been explained in detail elsewhere (Fernandez et al., 2003) a short summary is given here. All three samples per patient were treated equally. Therefore, a volume of 25 ll ejaculate or sperm suspension was mixed with liquid agarose (25 ll). This mix was pipetted onto precoated slides and covered with small coverslips. The slides were placed in a refrigerator (4C) for 5 min to allow the agarose to produce a microgel with the sperm cells embedded within. After gelation, coverslips were gently removed and the slides immediately immersed in an acid solution (7 min). Then, the slides were immersed in 10 ml of a lysing solution (25 min) since indirect proof of strand breaks requires denaturation of DNA. After a 5-minute washing step in distilled water, the probes were dehydrated in increasing concentrations of ethanol (70–90–100%) for 2 min each, air-dried and stored at room temperature. For light microscopy, slides were stained. Last but not least, the slides were briefly washed in distilled water and allowed to dry. Strong staining was preferred to easily visualize the periphery of the dispersed DNA halos (Figure 3). If possible, a minimum of 500 spermatozoa per sample were scored under the 100· objective of the microscope. Only sperm heads with a distinct halo have been reported to be without strand breaks. It is important to note that spermatozoa with a rather small halo have to be pooled with halo-negative spermatozoa (presumed DNA damage). A cut-off value of 18% was found above which a severe impact on further outcome is expected (Velez de la Calle et al., 2008).
40
Figure 3 Sperm chromatin dispersion (SCD) test in a patient showing approximately 30% strandbreak-positive spermatozoa in the raw semen. Arrow heads indicate spermatozoa with no or minor halos (i.e. those with strand breaks). Bar = 50 lm.
T Ebner et al. Percentage of strandbreak-positive spermatozoa in raw semen was found to be 15.8 ± 7.8% (range 5.0–42.1%). Treatment with density gradient marginally (14.2 ± 7.0%) reduced percentage of strandbreak-positive spermatozoa (range 2.0–30.9%). Processing the ejaculate with glass Zech-selector, however, completely eliminated strandbreak-positive spermatozoa in 17/19 cases (89.5%). Two ejaculates showed a minor percentage of affected spermatozoa after processing (<2.5%). Overall, the mean value of DNA damage after processing with the glass device was 0.4% ± 1.1% (range 0–2.5%) and differed significantly from density gradient (P < 0.001) and raw semen (P < 0.001). Using polyethylene Zech-selectors, in 16/21 cases (76.2%) no single spermatozoa with strand breaks was counted after sperm selection. The polyethylene device (0.5 ± 0.9%; range 0–3.0%) was significantly better in terms of strandbreak reduction as compared with density gradient (P < 0.001). There was no such difference between glass and plastic chambers. All five patients that had their ejaculate processed with both types of Zech-selectors did not show any strandbreak-positive spermatozoa after processing.
Results Discussion A total of 39 men participated in this prospective evaluation, which dealt with the efficiency of the Zech-selector in accumulating DNA-intact spermatozoa. Since two patients (5.1%) could not deliver semen on demand, the actual number of patients included was 37. The time of abstinence varied from 2 to 8 days (although 3–5 days were recommended). This period was neither related to the major sperm parameters nor to the percentage of spermatozoa with fragmented DNA. A mean ± SD of 4.4 ± 1.6 ml ejaculate was produced. The average concentration of spermatozoa was 76.5 ± 103.8 · 106/ml and the corresponding percentage of progressive motile spermatozoa (WHO grade a and b) was found to be 36.6 ± 19.4%. The factor most affected was sperm morphology with only 7.8 ± 6.9% normally formed spermatozoa on average. Sperm processing using the density gradient completely removed immotile spermatozoa and almost completely removed WHO grade c spermatozoa (about 1–2%). However, it did not select for morphologically normal spermatozoa. The Zech-selector performed significantly better in concentrating WHO grade a spermatozoa (P < 0.05) since all selected spermatozoa (100%) showed fast progressive motility. Within processing time, approximately 50% of the fast progressive spermatozoa were selected with the Zech-selectors. No such improvement in terms of sperm morphology was observed. Part of the ejaculate of 37 men was either referred to the glass chamber (n = 19) or the polyethylene device (n = 23). Due to a larger volume, five ejaculates were processed with both types of chambers. Since filling of the polyethylene selector was unsuccessful in two cases (8.7%), 35 patients could finally be included in the present study. Due to improper filling, 3/23 (13.0%) polyethylene Zech-selectors showed signs of contamination with immotile spermatozoa. Glass Zech-selectors never showed this problem.
Several factors are thought to induce DNA fragmentation, such as apoptosis, oxygen radicals, radio- and chemotherapy or environmental toxicants, to name but a few. Sakkas and Alvarez (2010) emphasized that DNA damage in spermatozoa can occur during production and/or transport of the male gametes. During spermatogenesis screening, mechanisms in the testes governed by Sertoli cells are responsible for the induction of apoptosis by marking individual spermatozoa with apoptotic markers, which in turn causes phagocytosis of these cells (Billig et al., 1996). Testicular endogenous nuclease activity, required for facilitating protamination, further increases the percentage of DNA-damaged spermatozoa (McPherson and Longo, 1993). It has been speculated that post-testicular sperm DNA fragmentation arising during sperm transport through the epididymis is even more frequent since testicular spermatozoa show lower levels of DNA damage than epididymal or ejaculated ones (Greco et al., 2005; Ollero et al., 2001). Immature spermatozoa (e.g. showing cytoplasmatic retentions) produce high concentrations of reactive oxygen species (ROS) and, as spermatozoa are highly packed in the epididymis, ROS-associated damage of DNA (either directly or via the activation of caspases) is very likely to occur (Desai et al., 2010; Ollero et al., 2001). On average, a 25% increase in seminal ROS concentration was found to be correlated with a 10% increase in DNA fragmentation (Mahfouz et al., in press). However, if testicular and epididymal mechanisms for removal of genomically defective spermatozoa do not work properly, a certain percentage of defective germ cells will later end up in the ejaculate presenting DNA strand breaks. The influence of such problems on reproductive outcome can only be estimated since it is not known to what degree the oocyte can compensate for a paternal factor. Even if a
Accumulation of DNA-strandbreak-free spermatozoa high percentage of spermatozoa in an ejaculate may have fragmented DNA, a non-affected spermatozoon could be chosen for intracytoplasmic sperm injection. This grey area could be one reason for the controversial discussion on the possible effect of sperm DNA strand breaks on further outcome (Borini et al., 2006; Bungum et al., 2007). Thus, both the actual extent of DNA damage in individual male gametes and the efficiency of certain sperm processing techniques to enrich DNA strandbreak-free spermatozoa are of clinical importance (Sakkas and Alvarez, 2010). Two major strategies for dealing with patients showing high rates of sperm DNA damage have been suggested (Sakkas and Alvarez, 2010): (i) use of testicular instead of ejaculated spermatozoa (Greco et al., 2005); and (ii) applying methods that help to accumulate unaffected ejaculated spermatozoa. As regards the latter approach, several technologies have been introduced. Magnetic cell sorting using annexin-V microbeads can effectively separate apoptotic and non-apoptotic spermatozoa (Said et al., 2005). Others reduced the percentage of spermatozoa with DNA strand breaks by either selecting based on maturity (Jakab et al., 2005), for example by the presence of hyaluronic acid bindingsites on the sperm head (Parmegiani et al., 2010a), or at higher magnification (Bartoov et al., 2003; Itzkan et al., 2007). As a common feature of all these methods, they can only reduce the percentage of spermatozoa revealing DNA damage but never completely eliminate them from further usage. According to Sakkas and Alvarez (2010), ‘the onus must now be shifted to identification of the DNA-damaged spermatozoa and how to select individual or populations of normal spermatozoa’. Appropriately enough, it could be demonstrated that the present Zech-selector is an efficient sperm processing technology in terms of exclusive selection of DNA-damage-free spermatozoa provided that chambers are not contaminated with native ejaculate during filling process. Since these selectors select good-quality spermatozoa (in terms of DNA fragmentation) without additional manipulation, e.g. centrifugation, it is very likely that no supplementary (artefactal) strand breaks will occur (Twigg et al., 1998; Donnelly et al., 2001). It seems to be important that sperm processing follows a strict schedule, since prolonged incubation at room temperature or at 37C after density gradient centrifugation may lead to an increase in strand breaks (Dalzell et al., 2004; Gosa ´lvez et al., 2009). In this respect, the swim-over period in the present set up was limited to 2 h, which favourably compares with data from cryopreserved spermatozoa (Gosa ´lvez et al., 2009). However, it has to be emphasized that the shorter the swim-over period is kept, the higher the chance to completely eliminate DNA-damaged spermatozoa from an ejaculate. Although suggested elsewhere (Ahmad et al., 2007; Jackson et al., 2010; Marchesi et al., 2010), the present study could not find a significant benefit of density gradient centrifugation on the accumulation of DNA-intact spermatozoa. According to Zini et al. (2000a,b), the reason for this observed phenomenon is related to the initial semen quality. It has been suggested that oligo- (Burrello et al., 2004; Høst et al., 1999), terato- (Høst et al., 1999; Muratori
41 et al., 2003), as well as asthenozoospermia (Giwercman et al., 2003; Irvine et al., 2000; Mahfouz et al., in press; Varghese et al., 2009) are associated with a higher percentage of sperm DNA aberrations. Based on the present data, motility is the sperm parameter that seems to be of utmost importance since the Zech-selector strictly separates spermatozoa according to their motility/velocity and not to their morphology (e.g. spermatozoa with smaller vacuoles, head deformations and/or cytoplasmic droplets passed the ejaculate-medium barrier). This is further supported by literature (Ramos and Wetzels, 2001; Van den Bergh et al., 1998) and also by the study centre’s observation that the upper-most spermatozoa (e.g. those swimming close to the surface of the supernatant) show significantly reduced rates of DNA fragmentation after density gradient centrifugation. In other words, simple overlaying of a sperm sample with medium (without centrifugation) could lead to similar results provided that swim-up time is limited to avoid mixture of both DNA-intact and affected spermatozoa. This would, at least in part, shed some light on the discussion whether DNA strandbreak measurements are predictive of treatment outcome or not (Alvarez and Lewis, 2008; Bungum et al., 2008), considering that Bungum et al. (2008) froze sperm samples immediately after density gradient treatment and did not allow for spatial separation of spermatozoa of highest motility. Failure in adequately separating progressive spermatozoa from slower ones is actually the major problem with all sperm processing techniques so far, since after a short period even the slowest spermatozoa are liberated from the pellet allowing for mixture of all types of motile spermatozoa, e.g. those with and without strand breaks. The biological answer to the question why fast progressive spermatozoa show no signs of DNA damage is interesting. Since both nuclear and mitochondrial DNA can be harmed by strand breaks (Alvarez, 2005), any impact on the latter type could cause alterations in ATP production which in turn is a prerequisite for optimal sperm motility. This is supported by the finding that sperm motility is directly related to the mitochondrial volume within the sperm midpiece. Mutations or deletions within mitochondrial DNA have also been associated with reduced sperm motility (for review, see Ozmen et al., 2007). Thus, it is most likely that WHO grade a spermatozoa are those spermatozoa that are neither harmed by nuclear or mitochondrial DNA damage. In the Zech-selector, obviously, slower spermatozoa (e.g. WHO grade b) cannot overcome the capillary gap between the outer and the inner ring due to their reduced energy and forward movement. An alternative explanation for the present results could be that the Zech-selector also selects for mature spermatozoa, which have been found to show less DNA damage (Parmegiani et al., 2010) and better motility (Yagci et al., 2008). This would be in line with recently published studies suggesting that intracytoplasmic sperm injection using presumed mature spermatozoa gives a better outcome (Paes Almeida Ferreira de Braga et al., 2009; Van den Bergh et al., 2009; Parmegiani et al., 2010a,b). It has to be admitted that, according to Sakkas and Alvarez (2010), SCD might not be the first choice for analysing DNA fragmentation due to its indirect approach. However,
42 it should not be forgotten that the same test has been used for all probes (native, density gradient and Zech-selector), thus any theoretical deviation in detection as well as misinterpretation of the data is minimized. Anyway, a prospective comparison between TUNEL and SCA methods on the same semen sample led Zhang et al. (2010) to state that ‘using bright-field microscopy, the SCD test appears to be more sensitive than the TUNEL assay.’ Chohan et al. (2006) concluded that SCSA, TUNEL and SCD predict the same levels of DNA fragmentation. To conclude, the present sperm processing technique guarantees exclusive accumulation of spermatozoa with fast progressive motility which were found to be those with intact DNA. Since there is evidence that spermatozoa with fragmented DNA might have higher rates of chromosomal aberration (Muriel et al., 2007), using the Zechselector could, theoretically, also reduce the percentage of aneuploidies in a processed sperm sample; however, this remains to be tested. A prospective, randomized, multicentre study is ongoing to evaluate the actual influence of sperm DNA damage on preimplantation development and pregnancy outcome.
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Accumulation of DNA-strandbreak-free spermatozoa Jakab, A., Sakkas, D., Delpiano, E., et al., 2005. Intracytoplasmic sperm injection: a novel selection method for sperm with normal frequency of chromosomal aneuploidies. Fertil. Steril. 84, 1665–1673. Mahfouz, R., Sharma, R., Thiyagarajan, A., et al., in press. Semen characteristics and sperm DNA fragmentation in infertile men with low and high levels of seminal reactive oxygen species. Fertil. Steril. doi:10.1016/j.fertnstert.2009.12.030 [Epub ahead of print]. Marchesi, D.E., Biederman, H., Ferrara, S., Hershlag, A., Feng, H.L., 2010. The effect of semen processing on sperm DNA integrity: comparison of two techniques using the novel Toluidine Blue Assay. Eur. J. Obstet. Gynecol. Reprod. Biol. 151, 176– 180. McPherson, S., Longo, F.J., 1993. Chromatin structure-function alterations during mammalian spermatogenesis: DNA nicking and repair in elongating spermatids. Eur. J. Histochem. 37, 109–128. Muratori, M., Maggi, M., Spinelli, S., Filimberti, E., Forti, G., Baldi, E., 2003. Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation. J. Androl. 24, 253–262. Muriel, L., Goyanes, V., Segrelles, E., Gosa ´lvez, J., Alvarez, J.G., Ferna ´ndez, J.L., 2007. Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis. J. Androl. 28, 38–49. Neguescu, A., Guillermet, C., Lorimier, P., Brambilla, E., Labat-Moleur, F., 1998. Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity. Biomed. Pharmacother. 52, 252–258. Ollero, M., Gil-Guzman, E., Lopez, M.C., et al., 2001. Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum. Reprod. 16, 1912–1921. Ozmen, B., Caglar, G.S., Koster, F., Schopper, B., Diedrich, K., Al-Hasani, S., 2007. Relationship between sperm DNA damage, induced acrosome reaction and viability in ICSI patients. Reprod. Biomed. Online 15, 208–214. Paes Almeida Ferreira de Braga, D., Iaconelli Jr., A., Ca ´ssia Sa ´vio de Figueira, R., Madaschi, C., Semia ˜o-Francisco, L., Borges Jr., E., 2009. Outcome of ICSI using zona pellucida-bound spermatozoa and conventionally selected spermatozoa. Reprod. Biomed. Online 19, 802–807. Parmegiani, L., Cognigni, G.E., Bernardi, S., Troilo, E., Ciampaglia, W., Filicori, M., 2010a. ‘Physiologic ICSI’: hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality. Fertil. Steril. 93, 598–604. Parmegiani, L., Cognigni, G.E., Ciampaglia, W., Pocognoli, P., Marchi, F., Filicori, M., 2010b. Efficiency of hyaluronic acid (HA) sperm selection. J. Assist. Reprod. Genet. 27, 13–16. Ramos, L., Wetzels, A.M., 2001. Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the TUNEL assay. Hum. Reprod. 16, 1703–1707. Said, T.M., Grunewald, S., Paasch, U., et al., 2005. Advantage of combining magnetic cell separation with sperm preparation techniques. Reprod. Biomed. Online 10, 740–746.
43 Sakkas, D., Alvarez, J.G., 2010. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertil. Steril. 93, 1027–1036. Seli, E., Gardner, D.K., Schoolcraft, W.B., Moffatt, O., Sakkas, D., 2004. Extent of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization. Fertil. Steril. 82, 378–383. Twigg, J.P., Irvine, D.S., Aitken, R.J., 1998. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum. Reprod. 13, 1864–1871. Van den Bergh, M., Emiliani, S., Biramane, J., Vannin, A.S., Englert, Y., 1998. A first prospective study of the individual straight line velocity of the spermatozoon and its influences on the fertilization rate after intracytoplasmic sperm injection. Hum. Reprod. 13, 3103–3107. Van Den Bergh, M.J., Fahy-Deshe, M., Hohl, M.K., 2009. Pronuclear zygote score following intracytoplasmic injection of hyaluronan-bound spermatozoa: a prospective randomized study. Reprod. Biomed. Online 19, 796–801. Varghese, A.C., Bragais, F.M., Mukhopadhyay, D., et al., 2009. Human sperm DNA integrity in normal and abnormal semen samples and its correlation with sperm characteristics. Andrologia 41, 207–215. Velez de la Calle, J.F., Muller, A., Walschaerts, M., et al., 2008. Sperm deoxyribonucleic acid fragmentation as assessed by the sperm chromatin dispersion test in assisted reproductive technology programs: results of a large prospective multicenter study. Fertil. Steril. 90, 1792–1799. Yagci, A., Ocali, O., Stronk, J., Murk, M., Huszar, G., 2008. Relationship between sperm-hyaluronic acid (HA) binding assay scores and HA-mediated increase in maintenance of sperm motility and velocity in IVF conditions. Fertil. Steril. 90, 215. World Health Organization, 1999. Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction, fourth ed. Cambridge University Press, Cambridge. Zhang, L.H., Qiu, Y., Wang, K.H., Wang, Q., Tao, G., Wang, L.G., 2010. Measurement of sperm DNA fragmentation using bright-field microscopy: comparison between sperm chromatin dispersion test and terminal uridine nick-end labelling assay. Fertil. Steril. 94, 1027–1032. Zini, A., Boman, J.M., Belzile, E., Ciampi, A., 2008. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: systematic review and meta-analysis. Hum. Reprod. 23, 2663–2668. Zini, A., Finelli, A., Phang, D., Jarvi, K., 2000a. Influence of semen processing technique on human sperm DNA integrity. Urology 56, 1081–1084. Zini, A., Nam, R.K., Mak, V., Phang, D., Jarvi, K., 2000b. Influence of initial semen quality on the integrity of human sperm DNA following semen processing. Fertil. Steril. 74, 824–827. Declaration: The authors report no financial or commercial conflicts of interest. Received 20 May 2010; refereed 3 September 2010; accepted 7 September 2010.
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ARTICLE
Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa T Ebner
a,*
, O Shebl a, M Moser a, RB Mayer a, W Arzt b, G Tews
a Landes- Frauen- und Kinderklinik, IVF-Unit, Linz, Austria; Institute of Prenatal Genetics, Austria
b
a
Landes- Frauen- und Kinderklinik,
* Corresponding author. E-mail address: [email protected] (T Ebner). Thomas Ebner, PhD, graduated with honours from the Paris Lodron University of Salzburg, Austria in 1992. After completing his doctorate and post-doctoral theses, he became a university lecturer in Salzburg. He has published more than 80 papers as first and co-author. Research interests include non-invasive IVF selection processes, laser application, vitrification and culture media. He was certified as a senior clinical embryologist in 2008. Currently he is scientific director of the European School of ART in Linz.
Abstract Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired
preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0–42.1%). Density gradient did not significantly improve the quality of spermatozoa selected (14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and, with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report on a sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: density gradient centrifugation, sperm chromatin dispersion test, sperm motility, strand breaks, Zech-selector
Introduction A relatively high number of patients fail to achieve pregnancy despite the obvious absence of a male or female
factor of infertility. It is likely that many of these couples actually present with a genomic male factor, including meiotic alterations, aneuploidy or sperm DNA damage (Sakkas and Alvarez, 2010). In particular, sperm DNA fragmentation
1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2010.09.004
38 is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome (Borini et al., 2006; Carrell et al., 2003; Duran et al., 2002; Evenson et al., 1999; Seli et al., 2004; Velez de la Calle et al., 2008; Zini et al., 2008). Several tests have been established for the analysis of sperm DNA fragmentation. Amongst others, TdTmediated-dUTP nick-end labelling (TUNEL) (Gorczyca et al., 1993), comet assay (Enciso et al., 2009; Hughes et al., 1996) and sperm chromatin structure assay (SCSA) (Evenson et al., 2002) as well as the sperm chromatin dispersion (SCD) test (Fernandez et al., 2003) are the approaches most commonly used in IVF laboratories. These assays can be subdivided into two categories, those directly detecting DNA damage (e.g. TUNEL) and those measuring DNA fragmentation after a rather mild denaturation process (e.g. SCSA, SCD). Although direct proof of strand breaks would be appreciated, all of the above mentioned tests unveil certain limitations (Bungum et al., 2004; Neguescu et al., 1998; Sakkas and Alvarez, 2010). One important aspect with respect to sperm DNA fragmentation is the question whether breaks are singleor double-stranded since single-stranded defects are probably easier to repair as compared with double-stranded DNA breaks (Sakkas and Alvarez, 2010). In this respect it should be kept in mind that the processing of spermatozoa could also cause an apparent increase in DNA strand breaks (Dalzell et al., 2004; Donnelly et al., 2001; Gosa ´lvez et al., 2009; Twigg et al., 1998); thus, the sperm processing technique applied for removing DNA-damaged spermatozoa is of utmost importance (Sakkas and Alvarez, 2010). Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa (Ahmad et al., 2007; Jackson et al., 2010; Marchesi et al., 2010), but, to date, there is no reliable approach to completely filter out spermatozoa with strand breaks from an ejaculate. This study was started in order to test the efficiency of a rather new sperm processing technique (Zech-selector) with respect to the reduction of spermatozoa with DNA damage.
Materials and methods During the study period, 39 patients with known male subfertility who presented at the study centre’s andrology laboratory for a second analysis of their ejaculate were recruited. The mean age of the men was 37.7 ± 6.5 years. An abstinence time of 3–5 days was recommended. All ejaculates were processed and analysed strictly according to the World Health Organization (WHO) manual (1999). Half of men suffered from isolated teratozoospermia (51%). A smaller percentage had isolated astheno- (8%) or oligozoospermia (8%). However, the remaining 33% of patients showed a drop in more than one sperm parameter, including five cases of oligoasthenoteratozoospermia (OAT). The study was approved by the Institutional Review Board of the Landes- Frauen- und Kinderklinik, Linz, Austria, and the patient received verbal information about the nature of the study. Participation in the study allowed the patients to receive the DNA analysis free of charge.
T Ebner et al. After control of liquefaction, the ejaculate was processed immediately in order to avoid excessive contact between seminal plasma and spermatozoa which could have altered chromatin packaging, thus possibly interfering with DNA staining. It was planned to make three analyses of DNA fragmentation per patient. After sterile masturbation, a small volume (about 25 ll) of raw semen was kept in order to have a reference value (sample 1). The rest of the ejaculate was split into two unequal parts in order to treat them differently. The first volume (1–2 ml) was processed using routine density gradient centrifugation technique (sample 2). In detail, semen was placed on the top of two layers (40% and 80%) of GM501 Gradient (Gynemed, Lensahn, Germany). After layering the sample was centrifuged at 180g for 20 min. Subsequently, both layers containing silane-coated colloidal silica were carefully removed and the pellet resuspended in BM1 medium (NMS Bio-Medical, Praroman, Switzerland). In order to reduce additional manipulation of the spermatozoa, only one centrifugation step was performed at 180g for 10 min. Finally, the purified sperm sample was incubated at 37C for approximately half an hour, to allow for swim-up, and then strandbreak measurement was performed. In parallel, a patented (European patent number 1,432,787) sperm selecting chamber (Zech-selector, AssTIC Medizintechnik GmbH, Leutsch, Austria) made of glass or polyethylene was filled with 1–3 ml of ejaculate (sample 3). These chambers accumulate an adequate number of motile spermatozoa without exposure to centrifugation stress (Ebner et al., 2003). In principle, both devices consist of two concentric wells which are overlaid by a U-ring and a cover glass (Figures 1 and 2) and progressive motile spermatozoa migrate from the ejaculate in the outer well to concentrate in the medium-filled inner well by using a capillary bridge created by the overlaying U-ring. After 1 h, a 25 ll sperm sample was taken from the central chamber and referred to further analysis. Patients whose ejaculate had to be processed for more than 1 h (e.g. due to delayed liquefaction) were excluded from the study for the sake of homogeneity of the study group. Thus, it could be guaranteed that all three samples (neat semen, density gradient and sperm selecting chamber) were analysed within 1 h (including time for liquefaction), in other words prolonged contact with seminal plasma was avoided. If the volume of ejaculate was large (>5 ml), both types of chambers (glass and polyethylene) were used giving four values in these patients. It has to be clarified that the only limitation with the present sperm preparation technique is that patients diagnosed with OAT were processed slightly differently. Since sperm count and progressive motility were reduced in these ejaculates, the number of spermatozoa migrating to the medium-filled inner well was reduced. Therefore, careful removal of the inner volume (in order not to cause contamination) and subsequent concentration of the motile spermatozoa by single-step centrifugation (10 min at 180g) was performed. It should be kept in mind that filling of the chambers can be tricky. It is important that a minimum volume of 2 ml is used for the polyethylene chamber and at least 3 ml should
Accumulation of DNA-strandbreak-free spermatozoa
a
39
b
Figure 1 Schematic cross-section of the sperm selector indicating the ejaculate in the outer ring (dots) and medium in the centre well (wave lines). Cover glass is not shown. (a) Sperm selector consists of the actual chamber (glass or polyethylene) and a U-ring (top left). (b) After filling of the chamber, the U-ring is inserted and creates a capillary bridge allowing motile spermatozoa to swim to the centre well (theoretical path of migration indicated by arrow).
Figure 2 The Zech-selector. The outer diameter of the glass chamber is 6.3 cm. The diameter of the inner circle, considered for concentration of motile spermatozoa, is 2 cm.
be filled in the glass chamber. If volume was less than required, the difference was filled up with BM1 medium. This worked without problems in the glass chamber, but in 2/23 (8.7%) polyethylene chambers, no contact between the ejaculate and the medium in the centre well could be achieved. This happened despite any possible drop in the volume of the polyethylene centre well being carefully placed. In addition, it turned out to be important to minimize movement of the U-ring once placed in order to avoid contamination of the inner well with immotile spermatozoa and other debris (due to suction forces). Theoretically, contamination can also occur if, after processing a certain volume of sperm suspension, is removed from the centre well for later analysis. In contrast to the polyethylene chambers (3/23 with contamination), the glass chambers (due to their construction) allowed for removal of the ejaculate from the outer ring, thus disconnecting the liquid bridge and avoiding suction forces. However, after a certain learning curve, filling of both chambers worked perfectly.
An improved SCD test, the so-called Halosperm assay (Halotech DNA SL, Tres Cantos, Spain), was used for the determination of the actual percentage of DNA-damaged spermatozoa. At the beginning of the study, processed sperm samples were also analysed with TUNEL assay. Since no difference in the percentage of DNA-fragmented spermatozoa was observed (Chohan et al., 2006), this additional evaluation was cancelled for financial reasons. Although the SCD test has been explained in detail elsewhere (Fernandez et al., 2003) a short summary is given here. All three samples per patient were treated equally. Therefore, a volume of 25 ll ejaculate or sperm suspension was mixed with liquid agarose (25 ll). This mix was pipetted onto precoated slides and covered with small coverslips. The slides were placed in a refrigerator (4C) for 5 min to allow the agarose to produce a microgel with the sperm cells embedded within. After gelation, coverslips were gently removed and the slides immediately immersed in an acid solution (7 min). Then, the slides were immersed in 10 ml of a lysing solution (25 min) since indirect proof of strand breaks requires denaturation of DNA. After a 5-minute washing step in distilled water, the probes were dehydrated in increasing concentrations of ethanol (70–90–100%) for 2 min each, air-dried and stored at room temperature. For light microscopy, slides were stained. Last but not least, the slides were briefly washed in distilled water and allowed to dry. Strong staining was preferred to easily visualize the periphery of the dispersed DNA halos (Figure 3). If possible, a minimum of 500 spermatozoa per sample were scored under the 100· objective of the microscope. Only sperm heads with a distinct halo have been reported to be without strand breaks. It is important to note that spermatozoa with a rather small halo have to be pooled with halo-negative spermatozoa (presumed DNA damage). A cut-off value of 18% was found above which a severe impact on further outcome is expected (Velez de la Calle et al., 2008).
40
Figure 3 Sperm chromatin dispersion (SCD) test in a patient showing approximately 30% strandbreak-positive spermatozoa in the raw semen. Arrow heads indicate spermatozoa with no or minor halos (i.e. those with strand breaks). Bar = 50 lm.
T Ebner et al. Percentage of strandbreak-positive spermatozoa in raw semen was found to be 15.8 ± 7.8% (range 5.0–42.1%). Treatment with density gradient marginally (14.2 ± 7.0%) reduced percentage of strandbreak-positive spermatozoa (range 2.0–30.9%). Processing the ejaculate with glass Zech-selector, however, completely eliminated strandbreak-positive spermatozoa in 17/19 cases (89.5%). Two ejaculates showed a minor percentage of affected spermatozoa after processing (<2.5%). Overall, the mean value of DNA damage after processing with the glass device was 0.4% ± 1.1% (range 0–2.5%) and differed significantly from density gradient (P < 0.001) and raw semen (P < 0.001). Using polyethylene Zech-selectors, in 16/21 cases (76.2%) no single spermatozoa with strand breaks was counted after sperm selection. The polyethylene device (0.5 ± 0.9%; range 0–3.0%) was significantly better in terms of strandbreak reduction as compared with density gradient (P < 0.001). There was no such difference between glass and plastic chambers. All five patients that had their ejaculate processed with both types of Zech-selectors did not show any strandbreak-positive spermatozoa after processing.
Results Discussion A total of 39 men participated in this prospective evaluation, which dealt with the efficiency of the Zech-selector in accumulating DNA-intact spermatozoa. Since two patients (5.1%) could not deliver semen on demand, the actual number of patients included was 37. The time of abstinence varied from 2 to 8 days (although 3–5 days were recommended). This period was neither related to the major sperm parameters nor to the percentage of spermatozoa with fragmented DNA. A mean ± SD of 4.4 ± 1.6 ml ejaculate was produced. The average concentration of spermatozoa was 76.5 ± 103.8 · 106/ml and the corresponding percentage of progressive motile spermatozoa (WHO grade a and b) was found to be 36.6 ± 19.4%. The factor most affected was sperm morphology with only 7.8 ± 6.9% normally formed spermatozoa on average. Sperm processing using the density gradient completely removed immotile spermatozoa and almost completely removed WHO grade c spermatozoa (about 1–2%). However, it did not select for morphologically normal spermatozoa. The Zech-selector performed significantly better in concentrating WHO grade a spermatozoa (P < 0.05) since all selected spermatozoa (100%) showed fast progressive motility. Within processing time, approximately 50% of the fast progressive spermatozoa were selected with the Zech-selectors. No such improvement in terms of sperm morphology was observed. Part of the ejaculate of 37 men was either referred to the glass chamber (n = 19) or the polyethylene device (n = 23). Due to a larger volume, five ejaculates were processed with both types of chambers. Since filling of the polyethylene selector was unsuccessful in two cases (8.7%), 35 patients could finally be included in the present study. Due to improper filling, 3/23 (13.0%) polyethylene Zech-selectors showed signs of contamination with immotile spermatozoa. Glass Zech-selectors never showed this problem.
Several factors are thought to induce DNA fragmentation, such as apoptosis, oxygen radicals, radio- and chemotherapy or environmental toxicants, to name but a few. Sakkas and Alvarez (2010) emphasized that DNA damage in spermatozoa can occur during production and/or transport of the male gametes. During spermatogenesis screening, mechanisms in the testes governed by Sertoli cells are responsible for the induction of apoptosis by marking individual spermatozoa with apoptotic markers, which in turn causes phagocytosis of these cells (Billig et al., 1996). Testicular endogenous nuclease activity, required for facilitating protamination, further increases the percentage of DNA-damaged spermatozoa (McPherson and Longo, 1993). It has been speculated that post-testicular sperm DNA fragmentation arising during sperm transport through the epididymis is even more frequent since testicular spermatozoa show lower levels of DNA damage than epididymal or ejaculated ones (Greco et al., 2005; Ollero et al., 2001). Immature spermatozoa (e.g. showing cytoplasmatic retentions) produce high concentrations of reactive oxygen species (ROS) and, as spermatozoa are highly packed in the epididymis, ROS-associated damage of DNA (either directly or via the activation of caspases) is very likely to occur (Desai et al., 2010; Ollero et al., 2001). On average, a 25% increase in seminal ROS concentration was found to be correlated with a 10% increase in DNA fragmentation (Mahfouz et al., in press). However, if testicular and epididymal mechanisms for removal of genomically defective spermatozoa do not work properly, a certain percentage of defective germ cells will later end up in the ejaculate presenting DNA strand breaks. The influence of such problems on reproductive outcome can only be estimated since it is not known to what degree the oocyte can compensate for a paternal factor. Even if a
Accumulation of DNA-strandbreak-free spermatozoa high percentage of spermatozoa in an ejaculate may have fragmented DNA, a non-affected spermatozoon could be chosen for intracytoplasmic sperm injection. This grey area could be one reason for the controversial discussion on the possible effect of sperm DNA strand breaks on further outcome (Borini et al., 2006; Bungum et al., 2007). Thus, both the actual extent of DNA damage in individual male gametes and the efficiency of certain sperm processing techniques to enrich DNA strandbreak-free spermatozoa are of clinical importance (Sakkas and Alvarez, 2010). Two major strategies for dealing with patients showing high rates of sperm DNA damage have been suggested (Sakkas and Alvarez, 2010): (i) use of testicular instead of ejaculated spermatozoa (Greco et al., 2005); and (ii) applying methods that help to accumulate unaffected ejaculated spermatozoa. As regards the latter approach, several technologies have been introduced. Magnetic cell sorting using annexin-V microbeads can effectively separate apoptotic and non-apoptotic spermatozoa (Said et al., 2005). Others reduced the percentage of spermatozoa with DNA strand breaks by either selecting based on maturity (Jakab et al., 2005), for example by the presence of hyaluronic acid bindingsites on the sperm head (Parmegiani et al., 2010a), or at higher magnification (Bartoov et al., 2003; Itzkan et al., 2007). As a common feature of all these methods, they can only reduce the percentage of spermatozoa revealing DNA damage but never completely eliminate them from further usage. According to Sakkas and Alvarez (2010), ‘the onus must now be shifted to identification of the DNA-damaged spermatozoa and how to select individual or populations of normal spermatozoa’. Appropriately enough, it could be demonstrated that the present Zech-selector is an efficient sperm processing technology in terms of exclusive selection of DNA-damage-free spermatozoa provided that chambers are not contaminated with native ejaculate during filling process. Since these selectors select good-quality spermatozoa (in terms of DNA fragmentation) without additional manipulation, e.g. centrifugation, it is very likely that no supplementary (artefactal) strand breaks will occur (Twigg et al., 1998; Donnelly et al., 2001). It seems to be important that sperm processing follows a strict schedule, since prolonged incubation at room temperature or at 37C after density gradient centrifugation may lead to an increase in strand breaks (Dalzell et al., 2004; Gosa ´lvez et al., 2009). In this respect, the swim-over period in the present set up was limited to 2 h, which favourably compares with data from cryopreserved spermatozoa (Gosa ´lvez et al., 2009). However, it has to be emphasized that the shorter the swim-over period is kept, the higher the chance to completely eliminate DNA-damaged spermatozoa from an ejaculate. Although suggested elsewhere (Ahmad et al., 2007; Jackson et al., 2010; Marchesi et al., 2010), the present study could not find a significant benefit of density gradient centrifugation on the accumulation of DNA-intact spermatozoa. According to Zini et al. (2000a,b), the reason for this observed phenomenon is related to the initial semen quality. It has been suggested that oligo- (Burrello et al., 2004; Høst et al., 1999), terato- (Høst et al., 1999; Muratori
41 et al., 2003), as well as asthenozoospermia (Giwercman et al., 2003; Irvine et al., 2000; Mahfouz et al., in press; Varghese et al., 2009) are associated with a higher percentage of sperm DNA aberrations. Based on the present data, motility is the sperm parameter that seems to be of utmost importance since the Zech-selector strictly separates spermatozoa according to their motility/velocity and not to their morphology (e.g. spermatozoa with smaller vacuoles, head deformations and/or cytoplasmic droplets passed the ejaculate-medium barrier). This is further supported by literature (Ramos and Wetzels, 2001; Van den Bergh et al., 1998) and also by the study centre’s observation that the upper-most spermatozoa (e.g. those swimming close to the surface of the supernatant) show significantly reduced rates of DNA fragmentation after density gradient centrifugation. In other words, simple overlaying of a sperm sample with medium (without centrifugation) could lead to similar results provided that swim-up time is limited to avoid mixture of both DNA-intact and affected spermatozoa. This would, at least in part, shed some light on the discussion whether DNA strandbreak measurements are predictive of treatment outcome or not (Alvarez and Lewis, 2008; Bungum et al., 2008), considering that Bungum et al. (2008) froze sperm samples immediately after density gradient treatment and did not allow for spatial separation of spermatozoa of highest motility. Failure in adequately separating progressive spermatozoa from slower ones is actually the major problem with all sperm processing techniques so far, since after a short period even the slowest spermatozoa are liberated from the pellet allowing for mixture of all types of motile spermatozoa, e.g. those with and without strand breaks. The biological answer to the question why fast progressive spermatozoa show no signs of DNA damage is interesting. Since both nuclear and mitochondrial DNA can be harmed by strand breaks (Alvarez, 2005), any impact on the latter type could cause alterations in ATP production which in turn is a prerequisite for optimal sperm motility. This is supported by the finding that sperm motility is directly related to the mitochondrial volume within the sperm midpiece. Mutations or deletions within mitochondrial DNA have also been associated with reduced sperm motility (for review, see Ozmen et al., 2007). Thus, it is most likely that WHO grade a spermatozoa are those spermatozoa that are neither harmed by nuclear or mitochondrial DNA damage. In the Zech-selector, obviously, slower spermatozoa (e.g. WHO grade b) cannot overcome the capillary gap between the outer and the inner ring due to their reduced energy and forward movement. An alternative explanation for the present results could be that the Zech-selector also selects for mature spermatozoa, which have been found to show less DNA damage (Parmegiani et al., 2010) and better motility (Yagci et al., 2008). This would be in line with recently published studies suggesting that intracytoplasmic sperm injection using presumed mature spermatozoa gives a better outcome (Paes Almeida Ferreira de Braga et al., 2009; Van den Bergh et al., 2009; Parmegiani et al., 2010a,b). It has to be admitted that, according to Sakkas and Alvarez (2010), SCD might not be the first choice for analysing DNA fragmentation due to its indirect approach. However,
42 it should not be forgotten that the same test has been used for all probes (native, density gradient and Zech-selector), thus any theoretical deviation in detection as well as misinterpretation of the data is minimized. Anyway, a prospective comparison between TUNEL and SCA methods on the same semen sample led Zhang et al. (2010) to state that ‘using bright-field microscopy, the SCD test appears to be more sensitive than the TUNEL assay.’ Chohan et al. (2006) concluded that SCSA, TUNEL and SCD predict the same levels of DNA fragmentation. To conclude, the present sperm processing technique guarantees exclusive accumulation of spermatozoa with fast progressive motility which were found to be those with intact DNA. Since there is evidence that spermatozoa with fragmented DNA might have higher rates of chromosomal aberration (Muriel et al., 2007), using the Zechselector could, theoretically, also reduce the percentage of aneuploidies in a processed sperm sample; however, this remains to be tested. A prospective, randomized, multicentre study is ongoing to evaluate the actual influence of sperm DNA damage on preimplantation development and pregnancy outcome.
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