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From the * Vincent Memorial Obstetrics &
Gynecology Service, Andrology Laboratory and In Vitro Fertilization Unit,
Massachusetts General Hospital, Boston, Massachusetts; the
Department of Environmental Health,
Occupational Health Program, Harvard School of Public Health, Boston,
Massachusetts; and the Department of
Bioengineering, University of Washington, Seattle, Washington.
| Correspondence to: Dr Russ Hauser, Department of Environmental Health, Occupational Health Program, Harvard School of Public Health, Building 1, Room 1405, 665 Huntington Ave, Boston, MA 02115 (e-mail: rhauser{at}hohp.harvard.edu). |
| Received for publication April 19, 2005; accepted for publication September 16, 2005. |
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Abstract |
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Key words: Semen quality, sperm motility, sperm morphology, DNA diffusion assay
Sperm DNA damage and sperm apoptosis have been considered as potentially useful indices of male fertility. Cellular apoptosis is a normal event that occurs both during and after embryonic development. Germ cell loss (Roosen-Runge, 1973; Allan et al, 1987), now recognized as occurring via apoptosis, is a dominant process during spermatogenesis and is regulated by p53, p21, caspaces, bcl-2, and Fas expression levels, with many alternate pathways (Lee et al, 1997; Sinha Hikim and Swerdloff, 1999; Print and Loveland, 2000; Martincic et al, 2001; Anzar et al, 2002; Said et al, 2004). It has been found that the number of sperm with Fas expression was low in subjects with normal sperm parameters but high in men with abnormal sperm parameters (Sakkas et al, 1999)
Mild to moderate genotoxic and cytotoxic insults to germ cells also induce apoptosis. Unlike somatic cells, it is unclear whether ejaculated sperm retain the capacity to undergo apoptosis (Sakkas et al, 1999; Wang et al, 2003: Lachaud et al, 2004). Male infertility appears to be positively correlated with increased levels of sperm with apoptotic markers (Oehninger et al, 2003; Paasch et al, 2003; Wang et al, 2003). The presence of immature sperm may contribute to increased levels of apoptotic markers in ejaculated sperm (Cayli et al, 2004). Although apoptosis is considered a mechanism to ensure selection of sperm cells with undamaged DNA, sperm with DNA damage that are not eliminated by apoptosis may fertilize an ovum (Sun et al, 1997; Morris et al, 2002).
Necrosis and apoptosis are 2 forms of cell death. In apoptotic cells, the plasma membranes are intact, whereas in necrotic cells, the plasma membranes lose their integrity and become leaky (Vermes et al, 1995). Apoptosis is physiologically programmed cell death that affects single cells without any associated inflammation in the surrounding tissues (Wyllie et al, 1980). Apoptosis is characterized by a distinct series of ultrastructural morphological (Wyllie et al, 1980) and biochemical changes (Wyllie et al, 1980; Williams and Smith, 1993). There is chromatin aggregation, cytoplasmic condensation, and indentation of nuclear and cytoplasmic membranes in apoptotic cells (Aznar et al, 2002). At the end of this process, the nucleus undergoes fragmentation, and the whole cell blebs and fragments into apoptotic bodies (Aznar et al, 2002).
While there have been many published studies investigating the relationship between traditional semen parameters and apoptosis, the results vary and firm conclusions are still outstanding (Sun et al, 1997; Gandini et al, 2000; Oosterhuis et al, 2000; Shen et al, 2002). Two commonly used methods for detecting spontaneous and induced apoptosis in sperm are the TUNEL assay (Baccetti et al, 1996; Weil et al, 1998; Gandini et al, 2000; Muratori et al, 2000; Oosterhuis et al, 2000; Hoyes et al, 2001; Koji, 2001; Ricci et al, 2002; Sakkas et al, 2002; Shen et al, 2002; Weng et al, 2002) and annexin V/PI staining (Hoyes et al, 2001; Ricci et al, 2002; Shen et al, 2002; Weng et al, 2002).
In the present study, we explored the associations between semen characteristics and the percent of apoptosis in ejaculated sperm in male partners of infertile couples. The DNA diffusion assay was used to assess sperm apoptosis, as it has been shown to be a simple and reproducible assay (Singh, 2000). This assay is based on the principle that apoptotic cells have numerous alkali-labile sites (Singh, 2000), and these sites, once exposed to alkaline conditions, yield low-molecular-weight DNA fragments. These pieces can easily diffuse in the agarose matrix giving the appearance of a hazy halo around the cell. This unique pattern of DNA gradient diffused in agarose is characteristic of apoptotic cells and is distinguishable from necrotic cells (Singh, 2000).
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Materials and Methods |
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Collection of Semen Samples
Semen was collected by masturbation into a sterile plastic specimen cup at
the hospital. Subjects were instructed to abstain from ejaculation for at
least 48 hours and no longer than 7 days before producing the semen sample.
The sample was liquefied for at least 20 minutes, but no longer than 1 hour,
before performing a routine semen analysis that included volume, pH, sperm
concentration, sperm motility, progressive motility, and sperm morphology. A
portion of the discarded semen was then used for the DNA diffusion assay. The
assay was performed on the discarded semen within 1.5 hours after receiving
the specimen.
Laboratory Evaluation
Concentration and Motility—
Semen samples were analyzed for sperm concentration and motion parameters
by computer-aided semen analyzer (CASA, HTM-IVOS Version 10; HTM-IVOS,
Beverly, Mass). Setting parameters and the definition of measured sperm motion
parameters for CASA were established by Hamilton Thorn Company. To measure
both sperm concentration and motility, 5 µL of semen from each sample was
placed into a prewarmed (37°C) Makler counting chamber (Sefi-Medical
Instruments, Haifa, Israel). A minimum of 200 sperm cells from at least 4
different fields was analyzed from each specimen. Motile sperm was defined as
World Health Organization (WHO) grade "a" sperm (rapidly
progressive with a velocity greater than or equal to 25 µm/s at 37°C)
plus "b" grade sperm (slow/sluggish progressive with a velocity
greater than or equal to 5 µm/s but less than 25 µm/s)
(WHO, 1999). The progressive
motile sperm was defined grade "a" sperm
(WHO, 1999).
Morphology— At least 2 slides were made for each fresh semen sample. The resulting thin smear was allowed to air dry for an hour before staining with a Diff-Quik staining kit (Dade Behring AG, Dudingen, Switzerland). Morphological assessment was performed with a Nikon microscope using 10x ocular lens combined with a 100x oil immersion objective lens (Nikon Company, Tokyo, Japan). A minimum of 200 sperm cells were counted from 2 slides for each specimen. Morphology was assessed using the strict criteria by Kruger et al (1988). The cutoff for morphology with normal or good pattern of fertilization is set at greater than or equal to 4%. Results were expressed as the percentage of normal spermatozoa and abnormal spermatozoa (head defects, midpiece defects, and tail defects). Semen analysis was performed before the DNA diffusion assay.
Endtz Test— The Endtz test is a rapid staining method for differentiation of polymorphonuclear leukocytes from germinal cells (Endtz, 1974). By adding a peroxidase solution to semen, a histochemical reaction occurs, staining the polymorphonuclear leukocytes a brown color. After staining, the specimen is read under microscopy in a Makler chamber and quantified by counting the number of polymorphonuclear leukocytes. Normal concentration of polymorphonuclear leukocytes in semen is less than 1 x 106/mL (WHO, 1999). A value greater than or equal to 1 million/mL is considered a positive test for polymorphonuclear leukocytes.
DNA Diffusion Assay— A small aliquot of the semen sample was analyzed for apoptosis using the DNA diffusion assay (Singh, 2000). Briefly, 60 mg of high resolution agarose 3:1 (Amresco, Solon, Ohio) was boiled in 9 mL of distilled water in a microwave oven, and 1 mL of 10x modified phosphate-buffered saline (PBS) was added to it (for one L: 80 g of NaCl, 2 g of KCl, 2 g of KH2PO4, 11.5 g of anhydrous Na2HPO4 or 29 g of Na2HPO4·7H2O, 32 g tris hydrochloride, pH 7.4). After adjusting volume to 10 mL by adding distilled water, the solution was boiled once more. The final concentration of agarose was 0.6%. The agarose was then dispensed in small aliquots and maintained at 55°C for 24 hours before use.
Preparing slides. Microgel electrophoresis (MGE) slides (Erie Scientific Co, Portsmouth, NH) were coated with 50 µL of 0.6% agarose 3:1. The first layer of the microgel was made by pipetting 50 µL of agarose on top of the frosted part of a slide while holding the slide horizontally in the left hand between thumb and index finger and smearing the agarose in one motion using a pipette tip held horizontally by the right hand. This layer was then air dried. The second layer of the microgel was made by mixing approximately 10 000 sperm cells in 5 µL of PBS with 50 µL of 0.6% agarose 3:1 for each slide. Fifty microliters of this mixture was layered onto the slides precoated with first layer of microgel, and this agarose cell mixture was immediately covered with a 24 x 50 mm2 #1 cover glass (Corning Glass Works, Corning, NY). The slides were put into a cold steel tray kept on ice. After removing the cover glass, 200 µL of 2% superfine resolution (SFR) agarose solution was layered as before to make a third layer of microgel. Use of 2% SFR agarose is essential for controlling the extent of DNA diffusion from apoptotic cells in agarose. The slide was covered with a cover slip after the third layer was made. After keeping the slides on ice for 2 minutes, cover glasses were removed and the slides were immersed and maintained for 10 minutes in a freshly made cold lysing solution (1.25 M NaCl, 1 mM tetrasodium EDTA, 5 mM Tris, 0.01% sodium lauryl sarcosine, 0.2% dimethylsulfoxide (DMSO) and 300 mM NaOH).
Neutralization, DNA precipitation, and staining of microgels. Slides for neutralization and DNA precipitation were immersed twice in freshly prepared 20 mM Tris, pH 7.4 in 50% ethanol with 1 mg/mL of spermine for 15 minutes. Slides were air dried. One slide at a time was stained with 50 µL 0.25 µM of Yellow-Orange Yellow-Orange-1 (YOYO-1) in 2.5% DMSO and 0.5% sucrose and immediately examined under a fluorescent microscope. Apoptotic cells were scored using a DMLB epifluorescence microscope (Leica, Wetzlar, Germany) having an excitation filter of 490 nm, a 500-nm dichroic filter, and an emission filter of 515 nm, emitting green fluorescence. One thousand cells were scored from each sample, and apoptotic cells were counted using 400x magnification. Sperm exhibiting diffuse halos in the DNA diffusion assay were considered apoptotic. The percentage of apoptotic cells with diffuse DNA and a hazy outline was calculated from a total of 1000 cells counted. The investigator who performed all apoptosis assessments was blinded to the results of the semen analyses.
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Statistical Analyses |
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Results |
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The results of the regression analyses exploring the relationship between percent apoptosis in ejaculated sperm and patient age and semen characteristics are presented in Table 3. In univariate analyses, 3 semen parameters were inversely associated with percent sperm apoptosis. After back transformation of the regression coefficients, there was -2.38% multiplicative change in apoptosis percent per 1 unit change in percent motility, (P = .0025), a -3.26% multiplicative change in apoptosis percent per 1 unit change in percent progressive motility (P = .0051) and a -10.44% multiplicative change in apoptosis percent per 1 unit change in percent normal morphology (P = .0045). Figures 1, 2, 3 show the relationships between these 3 parameters and sperm apoptosis. Sperm tail defects were positively associated with percent sperm apoptosis, there was an 8.83% multiplicative change in apoptosis percent per 1 unit change in percent tail defects (P = .0053). Typical halos are demonstrated in apoptotic human sperm cells (Figure 4).
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There were no statistically significant associations between percent sperm apoptosis and semen volume (P = .97), viscosity (P = .94), pH (P = .90), sperm concentration (P = .65), motile sperm concentration (P = .21), progressive motile sperm concentration (P = .20), total progressive motile sperm count per sample (P = .25), and percent abnormal morphology, head (P = .57), and midpiece (P = .09). There also was no statistically significant relationship between apoptosis and Endtz test results (P = .53). There was also no association between subject age and percent apoptosis (P = .78).
Nine men (39%) had above reference WHO value semen parameters (sperm concentration greater than 20 million/mL, motility greater than 50%, and progressive motility greater than 25%) and morphology with normal or good pattern of fertilization by Kruger strict criteria (percent normal morphology greater than 4%). Fourteen men (61%) had 1, 2, 3, or all 4 parameters below reference values. For example, some men had concentrations less than 20 million/mL, while others had motility or progressive motility less than 50% or 25%, respectively, or morphology less than 4%. The percent sperm apoptosis was lower (4.6%) in the group of men (n = 9) with all 4 semen parameters above reference values than it was in the group of men (n = 14) with 1 or more semen parameters below reference values (10.7%).
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Discussion |
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The DNA diffusion assay is a highly sensitive method for apoptosis assessment. Singh observed the sensitivity of the assay to be greater than 98% (Singh, 2000). The DNA diffusion assay has previously been used to assess sperm apoptosis (Singh et al, 2003), and results were well within the range observed by other commonly used assays such as TUNEL and annexin V. TUNEL assay, although sensitive, is associated with a number of artifacts, as it labels DNA strand breaks from any insult, in both apoptotic and nonapoptotic cells (Kocks et al 1998). Annexin V assay, based on detection of externalization of phosphatidylserine, may detect normal sperm after capacitation as apoptotic, thus overestimating total apoptotic cells (Kotwicka et al 2002).
The DNA diffusion assay is simple and specific for the detection of apoptosis and necrosis. Apoptotic cell nuclei display hazy halos while necrotic cell nuclei are larger and less defined, with halos that have clear outer borders (Singh, 2000).
In the present study, the mean (SD) percent apoptosis in ejaculated sperm was 8.3% (6.2), with a range from 1.1 to 20.1. The results are similar to those of Singh et al (2003), who showed 0.3% to 23% apoptosis of human sperm cells (mean, 6.5%) using the DNA diffusion assay. Similarly, using the TUNEL assay, Baccetti et al (1996) reported that 0.1% to 10% of sperm are apoptotic in human ejaculated sperm. Oosterhuis et al (2000) found that 20% of sperm from infertile men were TUNEL positive. Baccetti et al (1996) also reported a variance in the percentage of apoptotic sperm cells of approximately 0.1% in fertile controls, 10% in men with varicocele or infections (including AIDS), 20% in men with cryptorchidism, 25% in immature patients, and up to 50% in testicular seminoma carriers.
The present study found inverse associations between percent apoptosis in ejaculated human semen and sperm motility, progressive motility, and morphology. Similar findings were reported by several researchers (Sun et al, 1997; Barroso et al, 2000; Oosterhuis et al, 2000; Shen et al, 2002). In a pilot study, Oosterhuis et al (2000) found a significant inverse correlation between sperm motility and apoptosis by annexin V assay (r = -.289; P < .05) in ejaculated sperm. A cross-sectional observational study of 10 men from a fertility clinic conducted by Barroso et al (2000) using the gradient separation method reported that the semen fractions with low sperm motility had a significantly higher proportion of cells with DNA damage (P < .005) measured by TUNEL assay and monoclonal antibody labeling of single-stranded DNA. A report on 60 men from a fertility clinic in Singapore found that apoptotic cells (Annexin V-positive) were inversely correlated with sperm motility (Shen et al, 2002).
Some studies have been performed to link sperm morphology with the percentage of apoptotic sperm. Two studies found apoptosis positively associated with various forms of abnormal sperm morphology, including defects of the sperm head, midpiece, and tail (Gandini et al, 2000; Shen et al, 2002). Our data showed an inverse correlation between sperm apoptosis and normal or good pattern of fertilization morphology and a positive relationship between sperm tail defects and apoptosis.
In the present study, the percent of apoptosis was not significantly correlated with either sperm concentration or total sperm count. The literature to date has inconsistent results on the relationship between sperm apoptosis and measures of sperm quantity (Sun et al, 1997; Gandini et al, 2000; Oosterhuis et al, 2000; Shen et al, 2002). Oosterhuis et al (2000) found a strongly significant inverse correlation between the percentage of TUNEL-positive cells and the concentration of spermatozoa (r = -.629; P < .0001). They hypothesized that a low sperm concentration might be caused by a high rate of apoptosis. In a Canadian study of 298 men attending an infertility program, Sun et al (1997), using the TUNEL assay to assess DNA fragmentation, found a negative correlation with sperm concentration. In contrast, Shen et al (2002) in a study of 60 subfertile men reported that the percentage of apoptosis was significantly positively correlated with both sperm concentration and total sperm count when using the Annexin V and TUNEL assays. It is possible that these inconsistencies will be clarified as further studies are conducted.
Recently, using the DNA diffusion assay, Singh et al (2003) found an age-related decrease in the percent of sperm apoptosis among men of reproductive age. Sixty-six men were recruited; 40 were patients seeking an infertility evaluation, and 26 were controls. These subjects ranged in age from 20 to 57 years. Sperm exhibiting diffuse halos in the DNA diffusion assay were considered apoptotic. Overall, 6.5% ± 5.1% of sperm exhibited this pattern (range, 0.3% to 23%). The percent of apoptotic sperm was significantly inversely related to age (P = .029). The 2 older age groups (greater than 35 years and greater than 43 years) showed a highly significant decrease in apoptosis compared with the younger group (less than 25 years). However, there was no significant difference when subjects aged less than 25 years were compared with those aged greater than 25 years. In the present study, we did not find a relationship between age and apoptosis. This may be due to small sample size and narrow age range of our study population.
Percentages of apoptosis in human ejaculated sperm may correlate with fertilization rates after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) procedures (Sun et al, 1997; Levy et al, 2001; Wang et al, 2002). Detection of apoptosis in human sperm could provide additional information and may explain more about the causes of fertilization failures (Levy et al, 2001). In 143 IVF samples, a significant negative association was found between the percentage of sperm with DNA fragmentation, fertilization rate (P = .008), and embryo cleavage rate (P = .01) (Sun et al, 1997). Wang et al (2002) evaluated the relationship between sperm apoptosis and male infertility. They reported that sperm apoptosis by flow cytometry was significantly different between fertile and infertile groups, with results of 4.28 (±1.66)% and 18.67 (±8.55)%, respectively (P < .01).
We conclude that the use of the DNA diffusion assay to measure apoptosis in human ejaculated sperm in a clinical andrology laboratory is a simple and potentially useful assessment as part of an infertility evaluation. Our data showed significant relationships between apoptosis and sperm motility, progressive motility, morphology, and tail defects. Based on the findings of our study, additional study is warranted to confirm this association and to evaluate sperm apoptosis in relation to fertilization and pregnancy rates. We anticipate that the DNA diffusion assay for apoptosis in sperm may eventually be used as a diagnostic test, in conjunction with semen analysis, for male factor infertility.
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Acknowledgments |
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Footnotes |
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