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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Key words: chromatin structure/density gradient centrifugation/glass wool filtration/SCSA
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Introduction |
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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 < 0.010.001) 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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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 (0.62.5 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 23 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 (SD
t) and percentage of cells with denatured DNA (COMP
t) (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 COMP
t. 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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Results |
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Discussion |
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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, SD
t, and COMP
t) 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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Notes |
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References |
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Submitted on December 23, 1998; accepted on April 28, 1999.