Human Reproduction, Vol. 14, No. 8, 2015-2019,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Density gradient centrifugation and glass wool filtration of semen remove spermatozoa with damaged chromatin structure
1 3Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007 and 2 Human Reproduction Laboratory, Department of Obstetrics and Gynecology, University of South Dakota School of Medicine, Sioux Falls, SD 57105, USA
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Abstract |
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The ability of double-layered density gradient centrifugation (DGC) or glass wool filtration (GWF) of semen to remove spermatozoa with damaged chromatin structure was assessed by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility to sperm nuclear denaturation in situ. Ejaculates from 26 men attending a university-affiliated assisted reproduction laboratory were processed by DGC and GWF. Unprocessed, DGC- and GWF-processed specimens were assessed by the SCSA and by conventional semen parameters. Changes in chromatin structure were compared with conventional semen parameters. Both sperm preparation techniques yielded sperm suspensions with improved sperm chromatin structure as well as motility (%), forward progression (1–4) and viability (%). DGC was superior to GWF in the efficiency of recovering motile, morphologically normal, mature sperm suspensions. However, GWF produced improved chromatin integrity (SD
t) and viability. Moderate correlations between SCSA and conventional sperm parameters were observed. Nevertheless, the SCSA provides additional information about the biochemical integrity of sperm DNA and may be used in future studies to provide insight into assisted reproduction technology outcomes not explained by conventional sperm parameters.
Key words: chromatin structure/density gradient centrifugation/glass wool filtration/SCSA
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Introduction |
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In this era of assisted reproductive techniques, the integrity of sperm nuclear DNA is of paramount concern for the successful transmission of a competent paternal genome to the oocyte (Lopes et al., 1998
). The relationship between fertility and DNA integrity was studied by Evenson and co-workers, who showed that sperm chromatin structure, assessed by the sperm chromatin structure assay (SCSA), discriminated between fertile and subfertile men (P < 0.001; Evenson et al., 1999). Other researchers have shown that baseline sperm DNA damage (Kodama et al., 1997; Lopes et al., 1998) and susceptibility to oxidative and X-ray-induced DNA damage (Hughes et al., 1996) were greater in infertile men than in fertile controls. Therefore, sperm populations used for intracytoplasmic sperm injection (ICSI) may have a relatively high incidence of DNA fragmentation (Lopes et al., 1998). How this fragmentation will affect fertilization rates, embryo development and pregnancy outcomes remains unclear. For example, spermatozoa with exogenously induced oxidative DNA damage were still able to form pronuclei following ICSI (Twigg et al., 1998). However, other researchers have illustrated the importance of paternal influence in early embryo development (Janny and Ménézo, 1994) and have linked increased chromosomal breaks and acentric fragments in spermatozoa to repeated spontaneous abortions (Rosenbusch and Sterzik, 1991).
A negative correlation has been shown between the percentage of spermatozoa with DNA fragmentation after swim-up sperm preparation and ICSI fertilization rate (Lopes et al., 1998). This relationship between DNA fragmentation and fertilization failure indicates that technicians may not be able to distinguish between spermatozoa with normal and abnormal DNA based on conventional semen parameters. Therefore, decreasing the percentage of spermatozoa with damaged DNA by sperm preparation may be an important method for increasing ICSI fertilization above the current rate of 65% (Lopes et al., 1998). Although previous research has evaluated sperm preparation techniques [e.g. swim-up, density gradient centrifugation (DGC), glass wool filtration (GWF)] based on the ability to select motile (Gellert-Mortimer et al., 1988; LeLannou and Blanchard, 1988; McClure et al., 1989), morphologically normal (LeLannou and Blanchard, 1988; Menkveld et al., 1990; Vanderzwalmen et al., 1991, Yue et al., 1995; Yao et al., 1996) and mature spermatozoa (LeLannou and Blanchard, 1988; Colleu et al., 1996), little research has been undertaken that directly compares the improvement in chromatin integrity within a sample following density gradient centrifugation or glass wool filtration.
The SCSA is a favourable test to determine the effectiveness of sperm preparation techniques in selecting spermatozoa with adequate chromatin structure because it is rapid, relatively cost effective and statistically robust (Evenson et al., 1991). The SCSA determines the percentage of spermatozoa with abnormal chromatin structure, defined as susceptibility to acid-induced DNA denaturation in situ. SCSA data are strongly correlated (P < 0.01–0.001) with the level of DNA fragmentation assessed by BrdUTP incorporation and comets (single-cell electrophoresis) (Aravindan et al., 1997), indicating that susceptibility to in-situ DNA denaturation is an appropriate and accurate measure of sperm DNA integrity. Also, previous studies have indicated that SCSA parameters are independent of conventional semen parameters, providing additional information regarding the aetiology of male factor infertility (Evenson et al., 1991, 1999; Spano et al., 1998). Therefore, we utilized the SCSA to determine if either DGC or GWF yield sperm preparations with improved chromatin structure, and compared these changes in SCSA parameters with conventional sperm parameters.
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Materials and methods |
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Patients and initial analysis
Ejaculates from 26 men attending the Human Reproduction Laboratory at the University of South Dakota School of Medicine were obtained by masturbation after at least 36 h but not more than 4 days of abstinence. Semen samples were ejaculated into sterile polystyrene specimen containers and placed in a 37°C water-bath for 20–30 min to liquefy. After liquefaction, conventional semen parameters including volume (ml), concentration (x106/ml), motility (%), forward progression (1–4), morphology (% normal) and viability (% eosin-Y stained) were evaluated according to World Health Organization criteria (WHO, 1992). Aliquots of each semen sample were prepared for GWF and DGC semen preparation techniques to allow for direct pre- and post-processing comparisons of SCSA and conventional parameter data. The volume of semen prepared varied (ranging from 0.3 to 2.5 ml) between patients depending on the original ejaculate volume. A sub-sample from the unprocessed and GWF- and DGC-prepared spermatozoa was pipetted into cryovials and plunged into liquid nitrogen, where they were stored until thawed and analysed by flow cytometry.
Glass wool filtration (GWF)
Glass wool columns were prepared by gently inserting 30 mg of glass wool into the barrel of a 3 ml syringe, and compressed to a final thickness of 3 mm. The column was then rinsed with sperm washing media (SWM; Irvine Scientific, Santa Anna, CA, USA) until the filtrate, observed under microscopic examination, was free of glass wool fibres or no change was noted between washes. Prior to GWF, native semen was diluted with two volumes of SWM and mixed by pipetting gently up and down with a sterile transfer pipette. Following dilution, the semen suspension was centrifuged for 6 min at 300 g and resuspended in SWM. As previously described (Johnson et al., 1996), the washed sperm suspension was placed gently over the wet glass wool and allowed to filter by gravity. After the first three drops were discarded, the remaining filtrate was collected and analysed like the unprocessed (neat) sample for sperm concentration, motility, forward progression, morphology and viability. In addition, both the total number (total motile) and percentage of motile spermatozoa recovered (total motile recovered/total motile washed x100) in the filtrate were calculated.
Density gradient centrifugation (DGC)
Density gradients were prepared by pipetting 2.5 ml of 90% Enhance-S PlusTM (Conception Technologies, San Diego, CA, USA) into a sterile 15 ml conical centrifuge tube and overlaying 2.5 ml of 45% Enhance-S Plus. An aliquot (0.6–2.5 ml) of liquefied semen was placed onto the upper layer with a transfer pipette and centrifuged for 20 min at 300 g. The supernatant was aspirated and the pellet resuspended in 2–3 ml warmed SWM. The sample was centrifuged for 6 min at 300 g and the supernatant removed. The resuspension and centrifugation were repeated and the final pellet was resuspended in warmed (37°C) SWM. Conventional parameters of the DGC-prepared sperm suspension were assessed as the GWF-prepared filtrates.
Sperm chromatin structure assay (SCSA)
The SCSA determined the percentage of spermatozoa with abnormal chromatin structure in native semen and washed spermatozoa suspensions prepared by GWF and DGC. Abnormal chromatin structure was defined as increased susceptibility of DNA denaturation (Evenson et al., 1980). Amounts of DNA denaturation per cell were determined by flow cytometry which measured the shift of green (native DNA) to red (denatured, single-stranded DNA) fluorescence in acridine-orange stained nuclei. This shift is expressed as t (ratio of red fluorescence to red + green fluorescence). Populations with normal chromatin structure have a small standard deviation t (SDt) and percentage of cells with denatured DNA (COMPt) (Evenson and Jost, 1994). In addition, the SCSA identifies populations of spermatozoa with increased high green fluorescence (HGRN) which indicates the presence of immature spermatozoa with incomplete chromatin condensation.
Statistical analysis
Computer-generated means and standard deviations of red and green fluorescence and t (t = red/red + green fluorescence) values were analysed. The percentage of cells outside the main population of t (COMP) was obtained by delineation of the main population of t and extrapolating the right hand slope of the t frequency histogram to the x-axis (Figure 1). Data points to the left of this line are in the main population while those in the right are COMPt. Fluorescent debris, found near the origin of the cytogram, was in almost all cases easily delineated and excluded from analysis (Evenson et al., 1991).
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Analysis of variance (ANOVA) was used to compare differences in SCSA and conventional parameters by patient among unprocessed semen and GWF- and DGC-prepared spermatozoa suspensions. SCSA values were obtained by calculating the mean of two sub-samples taken from the same ejaculate and treatment. Regression analysis was used to determine the relationships among SCSA and conventional sperm parameters.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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SCSA variables
SCSA parameter (X
t, SDt, and COMPt) values indicative of DNA strand breaks (Aravindan et al., 1997) were significantly lower following either DGC or GWF (Table I and Figure 1
). GWF improved SDt more than DGC; however, Xt and COMPt did not vary by preparation method. The percentage of immature spermatozoa, identified by high green fluorescence (HGRN), was lower after DGC but was not significantly decreased following GWF (Table I and Figure 1).
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Conventional sperm parameters
Preparation by either DGC or GWF improved sperm motility, forward progression, and viability when compared to the unprocessed (neat) sample (Table II
). DGCprepared suspensions were superior to GWF-prepared suspensions in concentration, percentage of motile spermatozoa recovered and morphology. However, GWF yielded suspensions with a greater percentage of viable spermatozoa.
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Relationships between SCSA and conventional sperm parameters
There was at least a moderate, negative correlation between conventional (sperm concentration, motility, morphology and viability) and SCSA parameters (X
t, SDt and COMPt) in the neat, DGC-, and GWF-prepared sperm suspensions (Table III). Sperm viability following DGC was strongly correlated with Xt (–0.71, P < 0.001), SDt (–0.69, P < 0.001) and COMPt (–0.67, P < 0.001). In addition, a strong relationship existed between morphology of the neat suspensions and SDt (–0.74, P < 0.05). The percentage of immature spermatozoa (HGRN) was related to concentration of the neat, DGC, and GWF suspensions, morphology of the neat and DGC suspensions and motility of the GWF suspension.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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DGC and GWF significantly improved three SCSA parameters (X
t, SDt, and COMPt), indicating enrichment in chromatin integrity following either sperm preparation technique. GWF was superior to DGC in improving SDt. However, unlike GWF, DGC reduced the percentage of immature spermatozoa (HGRN) in the prepared sample. Elimination of immature spermatozoa may minimize oxidative stress resulting from exposure to concentrated enzymes found in unextruded cytoplasmic droplets of immature spermatozoa (Gomez et al., 1996). In future studies, it will be important to determine how these improvements in chromatin structure and maturity of the paternal genome will affect the viability of embryos, blastocysts and pregnancies. Improving embryo and blastocyst quality is critically important for assisted reproductive treatment success, where overall, <30% of fertilized human embryos implant after transfer to the uterus regardless of selection procedures (Edwards and Beard, 1999).
GWF and DGC effectively improved conventional semen parameters, including sperm motility, forward progression and viability relative to the neat sample. DGC yielded better efficiency of motile sperm recovery and improved morphology. Three SCSA parameters (Xt, SDt, and COMPt) were moderately correlated with conventional semen parameters including sperm morphology and motility, indicating that spermatozoa with the best physical and kinetic potential for fertilization also had superior chromatin integrity. Although these data indicated a stronger relationship between SCSA parameters and morphology than shown in previous studies (Evenson et al., 1991, 1999; Spano et al., 1998), only ~25% of the variation in chromatin integrity could be predicted based on morphology, indicating that adequate gross morphological and kinetic characteristics do not ensure the chromatin integrity of spermatozoa.
Future studies that determine how SCSA parameters of neat and prepared sperm affect assisted reproduction technique success may be used to determine which, if any, sperm preparation method compensates for abnormal chromatin in the unprocessed sample and should be used for specific assisted reproduction techniques.
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Acknowledgments |
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This work was supported in part by EPA Grant Number R820968–01, National Science Foundation Grants EHR-9108773, OSR-9452894 and the South Dakota Future Fund. This is South Dakota Agricultural Experiment Station Publication Number 3126 of the journal series.
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Notes |
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3 To whom correspondence should be addressed
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Submitted on December 23, 1998; accepted on April 28, 1999.
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