Hum. Reprod. Advance Access originally published online on November 1, 2006
Human Reproduction 2007 22(2):485-494; doi:10.1093/humrep/del415
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A new multiparameter flow cytometric method for human semen analysis
1 Clinical Analysis Unit, Department of Laboratory Medicine, Institute of Child Health IRCCS Burlo Garofolo and 2 Assisted Reproduction Unit, Department of Obstetrics and Gynaecology, Institute of Child Health IRCCS Burlo Garofolo and University of Trieste, Trieste, Italy
3 To whom correspondence should be addressed at: Assisted Reproduction Unit, Department of Obstetrics and Gynaecology, Institute of Child Health IRCCS Burlo Garofolo and University of Trieste, Via dell’Istria, 65/1, 34137 Trieste, Italy. E-mail: ricci{at}burlo.trieste.it
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BACKGROUND: The objectives of this study were (i) to evaluate whether the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) allows the detection of sperm apoptosis and (ii) to describe a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability and apoptosis as well as leukocyte concentration. METHODS: Semen samples from 68 patients were evaluated according to World Health Organization (WHO) criteria (normal, n = 26; abnormal, n = 42). The detection of activated caspases before and after betulinic acid (BA) incubation was carried out in 13 semen samples by flow cytometry using fluorescein-labelled inhibitors of caspases (FLICA). A multiparameter flow cytometric analysis was performed in 55 semen samples. Fluorescent microspheres were used to assess SC. Sperm apoptosis was detected by staining sperm with Syto 16 and 7-AAD. Leukocytes were counted using monoclonal anti-CD45. RESULTS: A significant correlation between the percentage of the spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found (r = 0.68, P = 0.0106; n =13). After incubation with BA, an increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods. There was a good correlation between flow cytometry and optical microscopy for sperm (r = 0.98, P < 0.0001) and leukocyte counting (r = 0.64, P <0.0001). The percentage of apoptotic sperm was inversely correlated with both SC (r = –0.303, P = 0.0246) and morphology (r = –0.384, P = 0.0050) but not with motility. CONCLUSIONS: The combination of Syto 16/7-AAD provides a sensitive assay to detect sperm apoptosis. The multiparameter flow cytometric method described offers the possibility of a simultaneous, simple, rapid and accurate assessment of several semen parameters.
Key words: apoptosis/flow cytometry/fluorospheres/sperm/Syto 16
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Cost-effective health care for the couple seeking help for infertility includes accurate semen analysis. Semen analysis performed with improper and therefore unreliable methods, without internal and external quality control, is a waste of resources and often leads to the wrong diagnosis, thus causing negative effects for both the couple and the health service provider. Guidelines for the standardization of the human semen examination have been developed by the World Health Organization (WHO) and are periodically reviewed (WHO, 1999
). However, a high inter-observer and intra-observer variability in basic semen analysis results has been described in many reports (Keel, 2004, for review). Various methods have been proposed to improve the reliability of semen analysis, such as a Coulter counter (Brotherton and Barnard, 1974), laser Doppler velocimetry (Brotherton, 1988) and computer-assisted semen analysis (CASA) (Boyers et al., 1989), but so far, none of these approaches have proved useful for routine application.
In recent years, flow cytometry has entered the andrology laboratory and has been extensively used to evaluate several different characteristics of sperm such as chromatin structure (Evenson et al., 1980; Spanò and Evenson, 1993; Pasteur et al., 1994), sperm viability (Evenson et al., 1982; Garner et al., 1994; Garner and Johnson, 1995; Ferrara et al., 1997), mitochondrial function (Evenson et al., 1982; Graham et al., 1990), acrosomal status (Graham et al., 1990; Thomas et al., 1997), antisperm antibodies (Haas and Cunningham, 1984; Rasanen et al., 1992; Ke et al., 1995) and spermatogenetic defects (Levek-Motola et al., 2005). The advantage of flow cytometry is that many thousands of cells can be analysed in a few seconds, giving a statistically more precise evaluation via a reproducible technique. Flow cytometric methods have been developed also for counting of sperm based on DNA staining (Spanò et al., 1984; Takacs et al., 1987) and on the ratio of cells to a known concentration of fluorospheres both in mammalian species (Evenson et al., 1993) and in human semen (Eustache et al., 2001).
In previous studies, we have shown the utility of flow cytometry to identify accurately the presence of leukocytes in a semen sample (Ricci et al., 2000) and to evaluate sperm viability and apoptosis (Ricci et al., 2002) using a combination of annexin V (AV) and propidium iodide (PI). The apoptosis is a common cell-death pathway, which is initiated by various different stimuli. Sakkas et al. (1999) showed that apoptosis is a major mechanism in regulating spermatogenesis in the human and that there are significant differences in molecular markers of apoptosis between males with normal and males with abnormal sperm parameters. Accurate detection of apoptotic cells is important for the determination of cell viability. The recognition of early apoptotic events would markedly improve reliability and convenience of apoptosis assays. The nucleic acid stain Syto 16 has been demonstrated to be able to distinguish apoptotic from non-apoptotic cells in several apoptosis models including cell lines and stem cells (Frey, 1995; Poot et al., 1997; Schuurhuis et al., 2001) and, recently, also in clinical samples (van der Pol et al., 2003). Apoptotic cells can be identified because they show a decreased (low) Syto 16 fluorescence, probably due to changes in their DNA structure (Frey, 1997; Poot et al., 1997). The combination of Syto 16 with a vital stain offers a sensitive, simple, inexpensive ‘live cell’ method for the discrimination of live, apoptotic and necrotic cells (van der Pol et al., 2003; Sparrow and Tippet, 2005). Viable cells can be identified because they show an intact (high) Syto 16 fluorescence. Necrotic cells can be identified by using 7-amino-actinomycin-D (7-AAD), a ready-to-use solution for the exclusion of non-viable cells in flow cytometric analysis. Like PI, 7-AAD penetrates only dead cells, but 7-AAD fluorescence is both less intense and of a longer wavelength (670 versus 610 nm for PI). These two properties make 7-AAD preferable as a viability marker when fluorescein isothiocyanate (FITC) and phycoerythrin (PE) are used to label surface antigens, being only a minimal spectral overlap between these emissions (Schmidt et al., 1992). It has been demonstrated that Syto 16 in combination with 7-AAD in a cell line model as well as in blood cells allows the identification of an early stage of apoptosis, not detected with Trypan Blue or 7-AAD alone or with conventional apoptosis tests, and only partly detected by the early apoptosis marker AV (Schuurhuis et al., 2001; Sparrow and Tippet, 2005). However, this method has never been tested on human semen. Several studies have demonstrated the existence of a caspase-dependant apoptotic pathway in ejaculated human spermatozoa (Said et al., 2004; Marchetti and Marchetti, 2005, for review). Activated caspases were detected in living spermatozoa by the carboxyfluorescein-labelled caspase inhibitor FAM-VAD-FMK (carboxyfluorescein, FAM, a derivative of benzyloxycarbonyl valylalanyl aspartic acid fluoromethylketone, z-VAD-FMK) (Paasch et al., 2003). It has been demonstrated that staining of spermatozoa with the FITC-Val-Ala-Asp-FMK (FITC-VAD-FMK) provides a valuable test assessing sperm apoptosis (Marchetti et al., 2004) and that caspase activation may be induced by specific agonists of apoptosis such as betulinic acid (BA) (Paasch et al., 2004; Grunewald et al., 2005a,b).
The objectives of the present study were (i) to demonstrate that Syto 16 is a sensitive probe to detect apoptosis in ejaculated spermatozoa, using the caspase activation assay as a comparison method and BA treatment as a confirmation method and (ii) to evaluate a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability, apoptosis and leukocyte concentration in human semen, using fluorospheres, Syto 16 and 7-AAD and antibodies anti-CD45.
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Patients
Semen samples were obtained from 68 patients [(mean (± SD) age 36.0 ± 5.9 years)] attending the Assisted Reproduction Unit of the Institute of Child Health IRCCS Burlo Garofolo, Trieste. All subjects were Caucasians and were the partners of women who failed to conceive after 24 months of unprotected intercourse. All subjects were asymptomatic for genitourinary infections. This study was approved by the Institutional Review Board of the Institute of Child Health IRCCS Burlo Garofolo. Informed consent for participation in the study was obtained from all patients.
Semen analysis
All semen samples were collected by masturbation into sterile containers after 3–4 days of sexual abstinence and were delivered to the laboratory within 45 min after ejaculation. Routine analysis was performed according to WHO guidelines (WHO, 1999). The variables taken into account were volume of ejaculate (ml), round cells concentration (x106/ml), SC (x106/ml), forward motility (%) and morphology (% of normal forms). A leukocyte count (x106/ml) was carried out by using standard peroxidase test. Following WHO criteria, semen samples were classified into normal (n = 26) or abnormal (n = 42).
Flow cytometric analysis of activated caspases
Levels of activated caspases were detected in sperm samples (n = 13) using fluorescein-labelled inhibitors of caspases (FLICA). This cell-permeable and non-cytotoxic caspase inhibitor binds covalently to active caspases. The fluorogenic substrate becomes fluorescent on cleavage by the caspases. The inhibitors were used with the appropriate controls according to the kit instructions provided by the manufacturers (Carboxyfluorescein FLICA Assay Kit, B-Bridge International, San Jose, CA, USA). A 150-fold stock solution of the inhibitor was prepared in dimethylsulphoxide (DMSO). It was further diluted in phosphate-buffered saline (PBS) to yield a 30-fold working solution. All test aliquots and controls (100 µl) were incubated at 37°C for 1 h with 10 ml of the working solution and subsequently washed with the rinse buffer.
Leukocytes separated from whole blood samples by density gradient centrifugation and incubated with BA (60 mg/ml) for 10 min served as positive control for caspase activation.
After labelling with FLICA, all samples were analysed directly by flow cytometry to detect caspase activity.
Induction of mitochondria-derived apoptosis
Mitochondria-derived apoptosis was induced as described by Grunewald et al. (2005a,b). Semen samples (n = 13) were washed in PBS by centrifugation at 400 g for 5 min. The supernatant was discarded, and the pellet was diluted in PBS at 
10 x 106/ml, and 1 ml was incubated with BA (Alexis, Gruenberg, Germany) at a final concentration 60 mg/ml for 10 min at room temperature to induce mitochondria-derived apoptosis (BA induced). An aliquot of the same sample in 1 ml of PBS was incubated under identical conditions and served as a negative control (non-induced). The samples were centrifuged at 400 g for 5 min, resuspended in 400 ml of PBS and prepared for the detection of caspases and for Syto-16 and 7-AAD staining.
Multiparameter semen analysis
Multiparameter flow cytometric analysis was performed on 55 semen samples from 55 consecutive patients (normal, n = 21; abnormal, n = 34). Syto 16 Green-Fluorescent nucleic acid stain from Molecular Probes (Eugene, OR, USA) 1 mM solution in DMSO was diluted 1:100 in PBS, aliquoted and stored at –20°C; 7-AAD (Via-Probe) solution ready to use was from BD PharMingen (San Diego, CA, USA). The source of PE and allophycocyanin (APC)-conjugated monoclonal anti-CD45 was Caltag Laboratories (Burlingame, CA, USA). Flow-Count fluorospheres for absolute count were from Beckmann-Coulter (Fullerton, CA, USA, lot 754863 at a concentration of 1016 beads/µl). One hundred microlitres of fresh semen specimen was stained for 20 min in the dark at room temperature using 2 µl of a 10-µM solution of Syto 16 (final concentration 200 nM), 10 µl of 7-AAD and 10 µl of monoclonal mouse anti-CD45 APC or anti-CD45 PE. The fluorospheres vial was gently mixed for 10–12 s, and 100 µl of Flow-CountTM fluorospheres was accurately pipetted (precision reverse pipetting with wet tip) before analysis. After the incubation period, 1 ml of PBS was added to each tube, and samples were analysed by flow cytometry.
Flow cytometric analysis was performed by using a FACSCalibur four-colour (Becton Dickinson, San Josè, CA, USA) equipped with a 488-nm argon laser with 530-nm (FL1), 585-nm (FL2) and 670-nm (FL3) band-pass fluorescence filters and a 635-nm red diode laser with a 661-nm band-pass filter (FL4). One hundred thousand events were collected in list mode and analysed with CELLQuest Pro software.
A gating strategy was used to allow the identification of live, dead and apoptotic sperm, as well as leukocytes and fluorospheres. A gate for improving the detection of the entire sperm population was determined on scattering measurements [forward-angle scatter (FSC) versus side-angle scatter (SSC)]. Because the gate for sperm on side-angle and forward-angle scatters could also include cells or debris with similar sizes and granularities as sperm (Figure 1B and D), the method based also on Syto 16 staining was applied (Figure 1A and C) to allow more precise identification of sperm population. A unique region R1 (Figure 1A and C) was set to include both Syto-16 low and Syto-16 high spermatozoa. Such a gated population was then analysed in another cytogram, Syto 16 versus 7-AAD (Figure 2), where the Syto 16 population displays different expression of 7-AAD. By using this gating strategy, it was possible to distinguish between viable, dead and apoptotic cells.
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A problem with multiparameter cytometric analysis is the possibility of fluorescence interference created by the dyes or fluorochromes that have close or overlapping emission spectra. Flow cytometry software has algorithms that can mathematically correct this spectral overlap; in terms of cytometry, this process is known as compensation. However, the choice of fluorochromes is important to make the compensation easy. The Syto 16 and 7-AAD combination has proved particularly suitable for cytometry analysis of sperm viability. In contrast to PI, the 7-AAD/DNA complex shows minimal spectral overlap with FL1 (green fluorescence) and FL2 (PE) emissions.
The fluorospheres contain a dye that has a fluorescence emission range of 525–700 nm and a dimension of 10 µm diameter. Two gates were set (R3 on FL1 versus SSC) and R4 on SSC versus FSC to identify accurately the fluorosphere population (Figure 1A and B). SCs (x106/ml) of the samples were calculated according to the following formula:
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where NS is the number of sperm counted, FC is the concentration of fluorospheres and NF is the number of fluorospheres counted. Concentration of fluorospheres indicates the number of fluorospheres per microlitre (known concentration), as given by the manufacturer, referred to the volume pipetted per sample. The method allows a count range of 10 000/ml up to a theoretical threshold of 1 000 000/ml spermatozoa.
Syto 16high/7-AADneg sperm were defined as viable, Syto 16low/7-AADneg sperm as apoptotic and Syto 16low/7-AADpos sperm as necrotic. The ratio between the Syto 16low/7-AADneg sperm and the total 7-AADneg sperm was defined as the apoptotic index.
Statistical analysis
Results were analysed using GraphPad Prism version 4.00 (GraphPad Software, San Diego, CA, USA). Correlations between the age of patients, routine semen analysis parameters and the results obtained by flow cytometry assays were computed using the Spearman’s rank correlation test. Comparison between normal and abnormal semen groups was performed using the Mann–Whitney U-test. Differences in percentage of apoptotic spermatozoa between age groups were determined using analysis of variance (ANOVA) because these data fulfilled assumptions for parametric testing.
All statistical tests were two-sided, and a P value of <0.05 was considered to be statistically significant.
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To assess whether Syto 16 might be able to detect apoptosis in ejaculated spermatozoa, we performed control experiments comparing the staining with Syto 16/7-AAD with the determination of activated caspases. A statistical significant positive correlation was found between the percentage of Syto 16low/7-AADneg spermatozoa and the percentage of spermatozoa containing activated caspases (r = 0.68, P = 0.0106) (Figure 3). Also, we used the Syto 16/7-AAD method to detect spermatozoa apoptosis in response to a pathological stimulus. After incubation with BA, a significant increase was observed both in the percentage of Syto 16low/7-AADneg spermatozoa (mean 14.4 ± 8.2 versus 28.8 ± 9.3%; P
0.0001) and in the percentage of spermatozoa containing activated caspases (mean 32.0 ± 11.8 versus 59.2 ± 7.5%; P 0.0001). An increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods (Figure 4).
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Dot-plot cytograms obtained with sperm sample, after staining with Syto 16, 7-AAD, PE and APC-conjugated anti-CD45, are shown in Figure 1, 2 and 5. The separation between debris and sperm (Figure 1A) was better evidenced by analysing the cytogram of SSC versus Syto 16 than through the analysis based only on SSC and FSC parameters (Figure 1B). The overlap of cellular debris (black) on sperm population (red) can invalidate the results of count and apoptosis. The effect of cellular debris on sperm analysis is studied in our previous work (Ricci et al., 2002
) in which we used the combination of a vital dye (6-CFDA) and PI to define sperm region accurately. However, this method cannot be used to assess different parameters in a single tube, because it is necessary to take into account the spectral emissions of various fluorescent probes. Our initial work was focused on the selection of a dye combination for use with the 488-nm standard laser of the FACSCalibur and a 635-nm red diode laser. It was observed that the Syto 16 dye resolved sperm population from debris and was also a sensitive probe to detect apoptosis; it emits on green fluorescence channel as FITC-AV and shows two peaks of staining corresponding to live cells (high) and apoptotic ones (low) (Figures 1A and 2). We found that 7-AAD is a very useful alternative to PI for its longer wavelength emission that makes compensation with FITC (green fluorescence or FL1) and PE (red fluorescence or FL2) easier. Therefore, the combination of Syto 16 with 7-AAD allowed the use of PE-conjugated anti-CD45 to simultaneously identify leukocytes (Figure 5C, region R3).
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We improved characterization of leukocytes using monoclonal anti-CD45 conjugated with APC, a dye that is excited by a red diode laser at 635 nm, with emission at 660 nm without overlapping with other dyes (Figure 5A, region R2).
There was a good correlation (r = 0.98, P < 0.0001) between flow cytometry and optical microscopy for sperm counting (Figure 6). White blood cell concentrations obtained via the peroxidase test and cytometry method using anti-CD45 were also significantly correlated (r = 0.64, P < 0.0001) (Figure 7).
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Table I summarizes the percentage of the cell populations identified using Syto 16 and 7-AAD. The percentage of apoptotic and necrotic sperm was significantly higher in the abnormal semen group than that in the normal semen group. However, there was an overlapping between the two groups (Figures 8–
10). The correlation between the semen parameters and the percentage of viable, apoptotic and necrotic cells is summarized in Table II. There was a significant correlation between the percentage of viable sperm and concentration and morphology. The percentage of apoptotic cells and the apoptotic index were inversely correlated with SC and morphology. The percentage of necrotic cells was inversely correlated only with the SC. No correlation was seen between patient’s age and leukocyte concentration and flow cytometry results. In sperm samples defined as normal by WHO (1999) criteria, no significant differences were detected in the percentage of apoptotic spermatozoa between the young patients (<33 years) and the older age groups (Table III).
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Discussion |
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Fluorocytometric methods based on the ratio of cells to a known concentration of fluorospheres to assess SC have been developed for mammalian (Evenson et al., 1993
) and, recently, for human semen (Eustache et al., 2001). However, the presence of other cells and debris in seminal fluid and the absence of specific markers for sperm cells make difficult the detection of sperm population. To solve these problems acridine orange (AO) or PI DNA sperm staining have been used (Evenson et al., 1993; Eustache et al., 2001), but permeabilization and staining with AO or PI do not allow the assessment of sperm viability. Therefore, other dyes have also been investigated, such as SYBR-14, a membrane-permeant nucleic acid stain that brightly stains the nuclei of living cells. The combination of SYBR-14 and PI was shown to be effective in differentiating between the living and the dead mammalian sperm (Garner et al., 1994; Hansen et al., 2002; Christensen et al., 2004).
In our study we used the fluorescent probe Syto 16 for DNA sperm staining. Syto16, like the fluorescent probe SYBR-14 stains the DNA of living and dead cells, but it is also able to distinguish apoptotic from nonapoptotic cells (van der Pol et al., 2003). In addition, Syto 16 proved to have a combination of favourable properties: extremely low intrinsic fluorescence, with quantum yields typically <0.01 when not bound to nucleic acids and >0.4 when bound to nucleic acids. Syto 16 can be excited at 488 nm and has large fluorescence enhancement on binding to nucleic acids, allowing the use of low, non-toxic (<10 nM) concentrations (Broxterman et al., 1997). We used 7-ADD to detect necrotic cells, because the PI staining technique showed toxic effects on viable cells (Bertuzzi et al., 1990). Furthermore, 7-ADD, like PI, penetrates only dead cells but its fluorescence is less intense and has a longer wavelength (670 versus 610 nm for PI).
The results of our present study suggest that Syto 16 can be considered as an apoptotic marker also for ejaculated spermatozoa. A significant correlation between the percentage of spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found. In addition, Syto 16 was able to detect also apoptosis induced by a pathological stimuli, such as BA incubation.
The results of our study also show that the flow cytometric method described in this article allows precise determination of SC. The correlation between the SC obtained by conventional optical microscopy and that by flow cytometry was very high, indicating that sperm counting by flow cytometry may be performed rapidly, reliably and objectively. Furthermore, this method offers several advantages over the previously reported methods.
By using only one tube, it is possible to evaluate simultaneously SC, viability and apoptosis, as well as leukocyte concentration.
No pretreatment of the semen sample is required. This avoids possible mistakes due to dilution, washing and centrifugation procedures. The acquisition of 100000 events per analysis provides a higher accuracy of parameters measurement.
Leukocyte concentration was assessed using APC-conjugated monoclonal anti-CD45. APC is excited by the second laser, so that it does not require compensation with Syto 16 and 7-ADD. Cytometers equipped with only one laser can be also used because the method is also optimized for use with PE-conjugated anti-CD45, which does not require a second laser.
The software that we used for the analysis was CellQuest from Becton Dickinson, but any software commercially available supplied with the cytometer was applicable for the determination of parameters of interest, using the gate strategy described. A critical passage in our method of analysis was setting correctly the regions around the populations of interest, creating a series of gates that would allow the operator to evaluate whether the results were coherent. For example, creating too large a region on sperm population identified by Syto 16 versus SSC, analysing apoptosis on cytogram displaying Syto 16 versus 7-AAD, the events corresponding to debris could fall into quadrant of Syto 16low/7-AADneg interfering with a correct evaluation of apoptosis rate. For this reason, the use of automated software to analyse regions is not advisable.
Finally, our protocol allows also simultaneous accurate assessment of sperm apoptosis.
In a previous work, we described a modified method to detect sperm apoptosis using AV and PI (Ricci et al., 2002). No significant differences in the percentage of apoptotic sperm between normal and abnormal semen subjects were observed, and we concluded that apoptosis, as it could be detected by AV, was not significantly correlated with semen abnormalities (Ricci et al., 2002).
In the present study, we analysed the correlations between semen parameters and apoptosis detected by a combination of Syto 16 and 7-AAD. We found a significant inverse correlation between the percentage of apoptotic cells and both SC and morphology. No correlation between sperm apoptosis and motility was found. This result could be explained hypothesizing that some apoptotic sperm were able to move and some others not. Alternatively, it can be hypothesized that in the first stage of apoptosis, the motility might not be affected. We have observed a significant difference between the normal and the abnormal semen group. In the abnormal semen group, the percentage of apoptotic sperm was twice as much as the normal semen group. However, there was an overlap between the two groups.
In ejaculates from subjects with normal sperm parameters, the percentage of apoptotic sperm was not significantly different between age groups. Therefore, Syto 16 spermatozoa staining cannot be considered as an event related to subject’s age.
We assume that in some cases the apoptosis increase could be the expression of spermatogenesis damage. In other cases, the apoptosis increase could be the effect of different factors, without significant changes in semen parameters. Likewise, not always spermatogenesis abnormalities probably have as consequence an increase of apoptosis (Weng et al., 2002). In our study, some of abnormal semen samples had a low percentage of apoptotic spermatozoa and a high percentage of necrotic spermatozoa (data not shown), suggesting that, in some cases, defective spermatogenesis may result in a necrosis sperm increase. Alternatively, in these subjects, the majority of apoptotic sperm may be in the later stage of apoptosis with a very high degree of membrane disorganization, and the 7-AAD passively enters the sperm cells. Moreover other studies yielded results that support our hypothesis (Paasch et al., 2003; Marchetti et al., 2004; Grunewald et al., 2005a,b). Indeed, a high proportion of sperm with activated caspases in samples from patients with normal sperm parameters (Marchetti et al., 2004) and in donors (Paasch et al., 2003; Grunewald et al., 2005a,b) has been reported. Further studies are required to assess, e.g. whether abnormal semen subjects with low apoptosis rate have better reproductive prognosis and whether normal semen subjects with high apoptosis rate have lower chance of achieving conception.
In conclusion, we observed that the combination of Syto 16/7-AAD provides a valuable assay for the detection of both spontaneous and induced sperm apoptosis. The multiparameter flow cytometric method presented here provides the possibility of a simultaneous, simple, rapid, reproducible and accurate assessment of several semen parameters. In addition, the method could easily be extended to the simultaneous investigation of other sperm characteristics such as mitochondrial function, acrosomal status or antisperm antibodies. Although costs for instrumentation are still significant, flow cytometry has so far proved a practicable solution for many laboratories because of its versatile employment in many fields of investigations.
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This work was supported by a grant from Ministero della Salute, Italia (Ricerca Finalizzata N. 139/01).
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References |
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Bertuzzi AD, Agnano I, Gandolfi A, Graziano A, Starace G, Ubezio P. (1990) Study of propidium iodide binding to DNA in intact cells by flow cytometry. Cell Biophys 17:257–267.[Web of Science][Medline]
Boyers SP, Davis RO, Katz DF. (1989) Automated semen analysis. Curr Prob Obstet Gynecol Fertil 12:165–200.
Brotherton J. (1988) Determination of human sperm count and sperm motility using a laser beam and the Doppler effect (LAZYMOT machine). Andrologia 20:33–43.[Web of Science][Medline]
Brotherton J and Barnard G. (1974) Estimation of number, mean size and size distribution of human spermatozoa in oligospermia using a Coulter counter. J Reprod Fertil 40:341–357.
Broxterman HJ, Schuurhuis GJ, Lankelma J, Oberink JW, Eekman CA, Claessen AM, Hoekman K, Poot M, Pinedo HM. (1997) Highly sensitive and specific detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain. Br J Cancer 76:1029–1034.[Web of Science][Medline]
Christensen P, Stenvang JP, Godfrey WL. (2004) A flow cytometric method for rapid determination of sperm concentration and viability in mammalian and avian semen. J Androl 25:255–264.
Eustache F, Jouannet P, Auger J. (2001) Evaluation of flow cytometric methods to measure human sperm concentration. J Androl 22:558–567.[Abstract]
Evenson DP, Darzynkievicz Z, Melamed MR. (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131–1133.
Evenson DP, Darzynkievicz Z, Melamed MR. (1982) Simultaneously measurement by flow cytometry of sperm cell viability and mitochondrial membrane potential related to cell motility. J Histochem Cytochem 30:279–280.[Web of Science][Medline]
Evenson DP, Parks JE, Kaproth MT, Jost LK. (1993) Rapid determination of sperm cell concentration in bovine semen by flow cytometry. J Dairy Sci 76:86–94.[Abstract]
Ferrara F, Daverio E, Mazzini G, Bonini P, Banfi G. (1997) Automation of human sperm cell analysis by flow cytometry. Clin Chem 43:801–807.
Frey T. (1995) Nucleic acid dyes for detection of apoptosis in live cells. Cytometry 21:265–274.[CrossRef][Web of Science][Medline]
Frey T. (1997) Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems. Cytometry 28:253–263.[CrossRef][Web of Science][Medline]
Garner DL and Johnson LA. (1995) Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 53:276–284.[Abstract]
Garner DL, Johnson LA, Yue ST, Roth BL, Haugland RP. (1994) Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. Biol Reprod 15:620–629.
Graham JK, Kunze E, Hammerstedt RH. (1990) Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry. Biol Reprod 43:55–64.[Abstract]
Grunewald S, Paasch U, Said TM, Sharma RK, Glander HJ, Agarwal A. (2005a) Caspase activation in human spermatozoa in response to physiological and pathological stimuli. Fertil Steril 83:Suppl 1, 1106–1112.
Grunewald S, Paasch U, Wuendrich K, Glander HJ. (2005b) Sperm caspases become more activated in infertility patients than in healthy donors during cryopreservation. Arch Androl 51:449–460.[CrossRef][Web of Science][Medline]
Haas GG and Cunningham ME. (1984) Identification of antibody laden sperm by cytofluorometry. Fertil Steril 42:606–613.[Web of Science][Medline]
Hansen C, Christensen P, Stryhn H, Hedeboe AM, Rode M, Boe-Hansen G. (2002) Validation of the FACSCount AF system for determination of sperm concentration in boar semen. Reprod Domest Anim 37:330–334.[CrossRef][Web of Science][Medline]
Ke RW, Dockter ME, Majumdar G, Buster JE, Carson SA. (1995) Flow cytometry provides rapid and highly accurate detection of antisperm antibodies. Fertil Steril 63:902–906.[Web of Science][Medline]
Keel BA. (2004) How reliable are results from the semen analysis? Fertil Steril 82:41–44.[CrossRef][Web of Science][Medline]
Levek-Motola N, Soffer Y, Shochat L, Raziel A, Lewin LM, Golan R. (2005) Flow cytometry of human semen: a preliminary study of a non-invasive method for the detection of spermatogenetic defects. Hum Reprod 20:3469–3475 [Epub 25 August 2005].
Marchetti C and Marchetti P. (2005) Detection of apoptotic markers in human ejaculated spermatozoa as new methods in human reproductive biology. Gynecol Obstet Fertil 33:669–677.[CrossRef][Medline]
Marchetti C, Gallego MA, Defossez A, Formstecher P, Marchetti P. (2004) Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters. Hum Reprod 19:1127–1134.
Paasch U, Grunewald S, Fitzl G, Glander HJ. (2003) Deterioration of plasma membrane is associated with activated caspases in human spermatozoa. J Androl 24:246–252.
Paasch U, Grunewald S, Dathe S, Glander HJ. (2004) Mitochondria of human spermatozoa are preferentially susceptible to apoptosis. Ann N Y Acad Sci 1030:403–409.[CrossRef][Web of Science][Medline]
Pasteur X, Metézéau P, Maubon I, Sabido O, Kiefer H. (1994) Identification of two human sperm populations using flow and image cytometry. Mol Reprod Dev 38:303–309.[CrossRef][Web of Science][Medline]
van der Pol MA, Broxterman HJ, Westra G, Ossenkoppele GJ, Schuurhuis GJ. (2003) Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis-corrected P-glycoprotein function in clinical samples. Cytometry B Clin Cytom 55:14–21.[Medline]
Poot M, Gibson LL, Singer VL. (1997) Detection of apoptosis in live cells by MitoTracker red CMXRos and SYTO dye flow cytometry. Cytometry 21:265–274.
Rasanen ML, Hovatta OL, Penttila IM, Agrawal YP. (1992) Detection and quantitation of sperm-bound antibodies by flow cytometry of human semen. J Androl 13:55–64.
Ricci G, Presani G, Guaschino S, Simeone R, Perticarari S. (2000) Leukocyte detection in human semen using flow cytometry. Hum Reprod 15:1329–1337.
Ricci G, Perticarari S, Fragonas E, Giolo E, Canova S, Pozzobon C, Guaschino S, Presani G. (2002) Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes. Hum Reprod 17:2665–2672.
Said TM, Paasch U, Glander HJ, Agarwal A. (2004) Role of caspases in male infertility. Hum Reprod Update 10:39–51.
Sakkas D, Mariethoz E, John JC St. (1999) Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Exp Cell Res 251:350–355.[CrossRef][Web of Science][Medline]
Schmidt I, Krall WJ, Uittenbogaart CH, Braun J, Giorgi JV. (1992) Dead cell discrimination with 7-amino actinomycin-D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13:204–208.[CrossRef][Web of Science][Medline]
Schuurhuis GJ, Muijen MM, Oberink JW, de Boer F, Ossenkoppele GJ, Broxterman HJ. (2001) Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen–thawed peripheral blood stem cell transplants. Bone Marrow Transplant 27:487–498.[CrossRef][Web of Science][Medline]
Spanò M and Evenson DP. (1993) Flow cytometric analysis for reproductive biology. Biol Cell 78:53–62.[CrossRef][Web of Science][Medline]
Spanò M, Calugi A, Capuano V, de Vita R, Gohde W, Hacker-Klom U, Maistro A, Mauro F, Otto F, Pacchierotti F, et al. (1984) Flow cytometry and sizing for routine andrological analysis. Andrologia 16:367–375.[Web of Science][Medline]
Sparrow RL and Tippett E. (2005) Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye. J Immunol Methods 305:173–187.[CrossRef][Web of Science][Medline]
Takacs T, Szollosi J, Balazs M, Gaspar R, Matyus L, Szabo G, Tron L, Resli I, Damjanovich S. (1987) Flow cytometric determination of sperm cell number in diluted bull semen by DNA staining method. Acta Biochim Biophys Hung 22:45–57.[Web of Science][Medline]
Thomas CA, Garner DL, DeJarnette JM, Marshall CE. (1997) Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa. Biol Reprod 56:991–998.[Abstract]
Weng SL, Taylor SL, Morshedi M, Schuffner A, Duran EH, Beebe S, Oehninger S. (2002) Caspase activity and apoptotic markers in ejaculated human sperm. Mol Hum Reprod 8:984–991.
World Health Organization. (1999) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction 4th (Cambridge University Press, Cambridge, UK).
Submitted on March 15, 2006; resubmitted on August 30, 2006; accepted on September 25, 2006.
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