Molecular Human Reproduction, Vol. 5, No. 1, 29-37,
January 1999
© 1999 European Society of Human Reproduction and Embryology
Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay
1 Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia, Via Anguillarese 301, 00060 Rome, and 2 Laboratory of Seminology and Reproductive Immunology, Department of Medical Pathophysiology, University of Rome `La Sapienza', 00100 Rome, Italy
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Abstract |
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The sperm chromatin structure assay (SCSA) is a flow cytometric (FCM) technique which exploits the metachromatic properties of Acridine Orange to monitor the susceptibility of sperm chromatin DNA to in-situ acid denaturation. SCSA was used to study the chromatin structure variations of human spermatozoa in semen, both before and after swim-up and after cryopreservation. Semen samples were provided by 19 healthy normozoospermic subjects attending pre-marriage checks. Each sample was divided into three aliquots: the first aliquot was evaluated without further treatment, the second underwent swim-up, and the third was stored according to standard cryopreservation techniques in liquid nitrogen at –196°C. Samples were also analysed by light and fluorescence microscopy (after Acridine Orange staining to evaluate the number of green fluorescent sperm heads), and by computer-assisted semen analysis. The results showed that post-rise spermatozoa represent a subpopulation characterized by a general improvement of the morphological (reduction of the percentage of abnormal forms and heads, increase of the green head sperm percentage) and kinetic parameters. This subpopulation also exhibited improved chromatin structure properties, confirming that these cells have the best structural and functional characteristics, indicative of optimal fertilizing ability. On the other hand, overall sperm quality deteriorates after cryopreservation. When thawed spermatozoa underwent an additional swim-up round, a general improvement of nuclear maturity was seen in the post-rise spermatozoa.
cryopreservation/flow cytometry/human spermatozoa/sperm chromatin structure/swim-up
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Introduction |
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The assessment of sperm chromatin quality in human semen samples is of relevance since chromatin abnormalities may be associated with failures in in-vitro assisted reproduction techniques (Bianchi et al., 1996
; Hoshi et al., 1996; Sakkas et al., 1996; Sun et al., 1997; Lopes et al., 1998). In addition, sperm cryopreservation is a necessary component of any assisted reproduction program. It is well known that temperature variations (e.g. during cooling, freezing and thawing) can cause detrimental changes in sperm structure at the membrane and mitochondrial levels, and so impair sperm function. It would be interesting to characterize the level of damage to the nuclear chromatin induced by extreme temperature changes. In this way, the sperm quality could be expressed in terms of cell-by-cell integrity, thus helping the physician to predict the chances of a successful fertilization. Moreover, the swim-up technique, together with the Percoll gradients and other cell separation methods, is a widely-used procedure to select a subpopulation of highly motile spermatozoa with alleged increased fertilizing ability. These sperm cells have an improved nuclear maturity, as assessed by microscopy (LeLannou and Blanchard, 1988; Colleu et al., 1996) or flow cytometric (FCM) techniques (Molina et al., 1995; Golan et al., 1997).
Since the proper functioning of spermatozoa seems to be related to chromatin integrity, methods focusing on the characterization of sperm chromatin condensation and stability have been receiving increasing attention. One of the most interesting techniques available today is represented by the sperm chromatin structure assay (SCSA), a FCM technique which exploits the metachromatic properties of Acridine Orange (AO) to monitor the susceptibility of sperm chromatin DNA to acid-induced denaturation in situ (Evenson et al., 1980,1991; Spanò and Evenson, 1993). Results are usually displayed as green versus red bivariate cytogram patterns and, from the limited applications reported in men so far (Evenson et al., 1980, 1991; Evenson and Melamed, 1983; Evenson and Jost, 1994; Golan et al., 1996, 1997; Fosså et al., 1997), there are clues that broadly heterogeneous SCSA patterns could be indicative of impaired fertility. Previous SCSA studies on spermatozoa obtained from proximal and distal epididymal locations in men (Golan et al., 1996), and also in other mammals (Evenson et al., 1989; Yossefi et al., 1994; Weissenberg et al., 1995), have demonstrated the usefulness of SCSA in identifying the sperm subpopulation able to complete the chromatin condensation process. It should not be overlooked that, in human samples, the relationship between conventional andrological parameters and SCSA results has been found to be very weak (Evenson et al., 1991; Fosså et al., 1997; Spanò et al., 1998). Therefore, SCSA parameters seem to reflect and display sperm characteristics complementary to, and beyond the resolution of, the light microscopy assessment.
In this study, SCSA has been used to evaluate the chromatin structure in fresh semen from 19 normozoospermic subjects and to measure nuclear maturity changes after swim-up or cryopreservation. The SCSA results were compared with those from standard microscopy assessment.
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Subjects
We studied 19 healthy subjects, aged 30–38 years, attending our Laboratory of Seminology and Immunology of Reproduction, Rome, Italy, for pre-marriage checks. All samples were considered normal according to World Health Organization guidelines (WHO, 1992). None of the subjects had received medical treatment in the 3 months prior to the study. All the subjects were counselled about the nature and purpose of the study and they signed a detailed consent form.
Semen evaluation
Semen was collected by masturbation after 3–5 days of abstinence. The ejaculates were allowed to liquefy at 37°C and examined within 1 h of collection. The parameters taken into consideration were volume of the ejaculate (ml), sperm concentration (n x 106/ml), forward motility (%), percentage of atypical forms (sperm and head morphology). The evaluation was carried out under a light microscope according to WHO (1992) criteria. All the seminal fluid examinations were performed by the same biologist (L.G.). Computer-assisted semen analysis (CASA) of sperm motility was also carried out. The CASA system (Cell Soft; Cryo Resources, New York, NY, USA) was equipped with a heated (35°C) stage. The parameters taken into consideration were curvilinear velocity (VEL, µm/s), linearity index (LIN), amplitude of lateral head displacement (ALH, µm), and beat cross frequency (BCF, Hz). For the CASA analysis, parameter settings were as follows: 25 frames to be analysed, 25 frames/s, 2 track points for calculation of motility, 8 track points for calculation of velocity, velocity range was 10–150 µm/s, cell size range was 4–20, 8 track points for calculation of ALH, minimum velocity for calculation of ALH was 20 µm/s, minimum linearity for calculation of ALH was 3.5 µm. At least 300 cells were examined in each sample. Each sample was divided into three aliquots: (A) the first aliquot was evaluated without further processing; (B) the second underwent swim-up; and (C) the third was stored according to standard cryopreservation techniques in liquid nitrogen at –196°C. In addition: (D) an aliquot of the cryopreserved sample underwent a further swim-up procedure after thawing. Each aliquot was analysed under the light microscope, by fluorescence microscopy after AO staining to evaluate the number of green fluorescent sperm heads, and finally, by FCM SCSA.
Swim-up procedure
We used the procedure described by Lopata et al. (1976). The seminal sample was centrifuged for 10 min at 300 g after dilution with Earle's solution. The supernatant was discarded and 0.5 ml of Earle's solution was layered onto the pellet. The spermatozoa were then allowed to migrate in the solution for 30 min at 37°C, in 5% CO2. This solution was then carefully collected and examined by light microscopy and CASA at the end of migration.
Semen cryopreservation
After liquefaction, an aliquot from each sample was diluted (1:1) with freezing medium (test yolk buffer; Irvine Scientific, Santa Ana, CA, USA). After an equilibration period at 37°C for 15 min, the mixture was aspirated into 0.25 ml straws, which were powder-sealed. The straws were frozen at –80°C in liquid nitrogen vapour for 8 min and then plunged into liquid nitrogen (–196°C) for storage. Finally, the semen was removed from the liquid nitrogen, thawed at room temperature for 15 min and examined by light microscopy and CASA system.
Swim-up after cryopreservation
After thawing, the semen aliquots were diluted with Earle's solution (1:2) and centrifuged for 10 min at 300 g. The supernatant was discarded and 0.5 ml of Earle's solution was layered on the pellet. The spermatozoa were then allowed to migrate in the solution for 30 min at 37°C, in 5% CO2. This solution was then carefully collected and examined by light microscopy and CASA after migration.
Acridine Orange test
The spermatozoa were washed twice in a salt solution, smeared on slide, air-dried, fixed overnight in Carnoy's solution (3:1, methanol:glacial acetic acid) and air-dried again. Slides were dipped in a 0.1 M citric acid solution (pH 2.5) for 5 min at room temperature and rinsed several times with distilled water. The slides were finally stained with an AO solution (0.2 mg/ml in water). After 5 min, each smear was washed with distilled water and covered with a coverslip and sealed with nail polish to prevent the smears from drying. Smears were examined using a fluorescence microscope (Leica, Dialux 22, Darmstadt, Germany) with a 490 nm excitation light and a 530 nm barrier filter. Nuclei from ~500 spermatozoa were examined and scored as fluorescing green or red. The evaluation of each microscopic field did not exceed 40 s to minimize photobleaching effects.
Flow cytometric analysis of sperm chromatin structure (SCSA)
We used the SCSA procedure described by Evenson and Jost (1994), with minor modifications. Samples were diluted with TNE buffer (0.15 M NaCl, 0.01 M Tris–HCl, 1 mM EDTA, pH 7.4) containing 10% glycerol into 2 ml Eppendorf snap cap tubes at a final sperm concentration of 2x106/ml. The tubes kept on dry ice until they were transferred to an ultra-cold freezer (–80°C), and stored there until FCM analysis. All these samples were coded and the FCM measurements were carried out blindly. After thawing on crushed ice, sperm cells were subjected to partial denaturation of DNA in situ and then stained with AO. 0.2 ml aliquots were admixed with 0.4 ml of a low pH detergent solution (0.17% Triton X-100, 0.15 M NaCl, and 0.08 N HCl, pH 1.4). After 30 s, the cells were stained by adding 1.2 ml of a solution (0.1 M citric acid, 0.2 M Na2HPO4, 1mM EDTA, 0.15 M NaCl, pH 6.0) containing 6 mg/l of chromatographically-purified AO (Molecular Probes, Eugene, OR, USA). Cells were analysed by a Facstar Plus flow cytometer (Becton Dickinson, San Josè, CA, USA), equipped with a 6W Ar ion laser (Innova 306, Coherent, Santa Clara, CA, USA), tuned at 488 nm and operated at a power output of 300 mW, light mode. When excited with a blue light source, AO intercalated to double-stranded DNA fluoresces green (530 ± 30 nm) and AO associated with single-stranded DNA fluoresces red (>630 nm). Since the residual RNA molecules in the mature spermatozoon do not interfere with the measurement (Evenson et al., 1985), the ratio of red to green fluorescence reflects the presence of single versus double-stranded DNA. A total of 5000 cells were measured for each sample. All measurements began 3 min after AO staining with a flow rate of ~200 cells/s. For instrument set-up and calibration we used aliquots from a normal human ejaculate sample retrieved from our laboratory repository. Calibration aliquots were thawed and measured at each start-up of the flow cytometer and after every 10 samples to ensure stability of the instrument from sample to sample. Scattergram analysis on raw data, with each point representing the coordinate of red and green fluorescence intensity values for every individual spermatozoon, was carried out using Becton Dickinson standard software. Events accumulated in the lower left corner correspond to sample debris and were excluded from the analysis. The bivariate data can be conveniently expressed by the function alpha T (
T) which is the ratio of red to total (red plus green) fluorescence intensity (Darzynkiewicz et al., 1975), thus representing the amount of denatured, single-stranded DNA over the total cellular DNA. T was calculated (ListView; Phoenix Flow Systems, San Diego, CA, USA) for each sperm cell in a sample and the results were expressed as the mean (X T), the standard deviation (SD T) of the T distribution, and as the percentage of cells with high T values, usually called Cells Outside the Main Population (COMP T), representing the cells with an excess of single-stranded DNA. T can range between 0 and 1 but, for practical considerations, it is generally and conveniently reported with values spanning between 0 and 1024 channels of fluorescence. Additionally, other parameters considered in the SCSA analysis are the mean (X Green) and standard deviation (SD Green) of the green fluorescence intensity distribution. The green fluorescence intensity distribution reflects the amount of the residual double-stranded DNA after the acidic treatment and AO accessibility depends on the correct protamine deposition process. Under these assumptions, spermatozoa with structurally altered chromatin are expected to elicit an increased proportion of single-stranded DNA (that is, higher values for the T parameters) and/or higher values for the green fluorescence parameters (i.e. anomalies in the histone to protamine exchange). The code was revealed only after completion of all the FCM SCSA measurements.
Statistical analysis
Statistical analyses were carried out using the SPSS software (release 7.0) for Windows 95 (SPSS Inc., Chicago, IL, USA). Analysis of variance for repeated measures was used to assess the overall significance, using the `within-subjects factor' method. The Greenhouse–Geisser correction of degrees of freedom was applied in order to better control any type-I errors. This analysis was repeated for each of the microscopy- and SCSA-derived parameters. Whenever P < 0.05, planned comparisons between the four types of sperm aliquots were performed in order to determine which class was significantly different.
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Microscopy
The mean values (±SD) of the fresh semen samples were as follows: volume 3.8 ± 1.1 ml (range 3.0–7.0 ml), sperm concentration 76.4 ± 43.7x106 spermatozoa/ml (range 28–180x106), forward motility 54.7 ± 8.6% (range 35–65%), total atypical forms 44.4 ± 8.6% (range 36–68%), head atypical forms 32.9 ± 8.7% (range 20–52%). The kinetic parameters values evaluated by the CASA system were as follows: VEL 58.5 ± 3.6 µm/s (range 52.8–65.3 µm/s), LIN 6.1 ± 0.3 (range 5.4–6.6), ALH 3.9 ± 0.4 µm (range 3.0–4.4 µm), BCF 13.9 ± 0.3 Hz (range 13.2–14.3 Hz). The percentage of green fluorescent heads after AO staining was 71.7 ± 7.7% (range 59–89%). The descriptive statistics of the sperm parameters evaluated microscopically for swim-up, cryopreserved, and post-rise spermatozoa after cryopreservation samples is shown in Table I
. All variables evaluated in the samples that underwent swim-up were significantly different from the corresponding fresh samples. The percentage of cells with forward motility increased to 87.1 ± 4.5%, the frequency of abnormal forms decreased to 24.8 ± 4.7%, the percentage of abnormal heads dropped to 20.8 ± 4.6%, and the percentage of green fluorescent sperm heads increased up to 83.6 ± 3.4%. These same variables differed significantly also in cryopreserved samples, the only exception being the CASA-derived parameter ALH. The post-rise spermatozoa from cryopreserved samples also showed a clear improvement of the associated variables when compared with the cryopreserved samples. Consequently, the comparison between the post-rise and swim-up samples, reflecting the properties of the swim-up selected spermatozoa, is also significantly different except for the CASA-derived parameters LIN and BCF. Altogether, these results indicate a general deterioration of semen quality due to cryopreservation, whereas post-rise spermatozoa represent a subpopulation of cells characterized by an improvement of kinetic parameters, by an enhanced percentage of green fluorescent heads and by a reduction of morphologically abnormal forms.
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Flow cytometry
A representative FCM fluorescence bivariate cytogram together with the corresponding
T and green fluorescence distributions, obtained after SCSA analysis of one fresh semen sample, is shown in Figure 1. The fraction of cells with abnormal chromatin (COMP T) is boxed off in the cytogram and in the corresponding frequency histograms. Bivariate cytograms and the corresponding T and green fluorescence distributions, obtained after SCSA analysis of the same sample in the four different experimental conditions are shown in Figure 2. It is evident that the COMP T is markedly reduced after swim-up in comparison with the corresponding unselected samples. However, the cytogram patterns look similar for both the fresh and cryopreserved samples. These results were consistently found for all the 19 ejaculates, regardless of the pattern variability in their respective SCSA bivariate cytograms. The numerical results of the SCSA assessment are also shown in Table I
. The mean values (±SD) calculated on fresh, unselected samples were: 215.5 ± 12.5 channels (range 201.7–246.3) forxT, 66.1 ± 16.4 channels (ranges 44.3–101.1) for SD T, 12.1 ± 7.9% (range 4.5–40.1%) for COMP T, 451.1 ± 30.1 channels for X Green (range 416.3–549.1), and 99.5 ± 17.0 channels for SD Green (range 77.7–114.2). The SCSA parameters differed significantly in samples that underwent cryopreservation, with the exception of SD T. The increase in the frequency of COMP T cells, together with the shift of the green fluorescent distribution parameters, can be indicative of the physical stresses the cryopreserved samples have experienced leading to chromatin deterioration in some of the spermatozoa present in the native sample. On the other hand, by and large, the population of migrated spermatozoa (swim-up and post-rise) exhibited a general improvement of all SCSA-related parameters (except SD T), when compared with the unselected populations. However, the fraction of COMP T cells in the post-rise samples was not markedly different from the unselected cryopreserved samples but remained significantly higher than that of post-rise spermatozoa from fresh semen. This picture probably reflects the fact that the highly motile cells selected from cryopreserved samples still suffer from residual chromatin damage induced by the deep freezing. The subpopulations of highly motile cells, looking also at the dispersion (i.e. SD) of the various SCSA parameters around their mean value, shows superior and more homogeneous chromatin structure characteristics than that of unselected fresh or cryopreserved semen samples. Therefore, the swim-up procedure represents a positive selection for the highly motile cell fraction which, in turn, is associated to improved chromatin structure. The fraction of COMP T cells, calculated on each of the 19 samples in the four different experimental conditions, is reported in Figure 3.
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Discussion |
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The packaging of chromatin in its final form into the sperm nucleus is a long and complex process starting in the very early stages of the spermiohistogenesis when histones are replaced firstly by transition proteins and finally by protamines (Meistrich, 1993
; Banerjee et al., 1995; Kramer and Krawetz, 1997). Additional steps occur after ejaculation, with the participation of seminal plasma, in the evolution of chromatin stability, a pathway culminating in a smaller mature sperm cell which requires less energy to support motility and ready to fertilize. Packaging of DNA into maximally condensed chromatin is associated with the disulfide cross-linkages established between cysteine molecules of protamines. The progressive sperm chromatin packaging seems to be linked to a complex process of DNA cutting and ligating (Sakkas et al., 1995). The presence of naturally occurring DNA strand breaks in mature human spermatozoa has been measured by in-situ nick translation (Bianchi et al., 1993), in-situ terminal deoxynucleotidyl transferase driven UTP nick-end labelling (TUNEL assay) (Gorczyka et al., 1993; Sailer et al., 1995; Aravindan et al., 1997), and single cell gel electrophoresis (COMET assay) (Hughes et al, 1996; Anderson et al., 1997; Aravindan et al., 1997). In particular, in human sperm samples, it has recently been demonstrated that DNA strand breaks identified by the COMET assay are strongly correlated with the frequency of COMP T cells as evaluated by the SCSA (Aravindan et al., 1997). Thus, in our experimental setting, the T parameters appear to be linked, most probably, to chromatin alterations associated with DNA nicks. On the other hand, an increased intensity of green fluorescence is likely to be due to increased native, non-denatured DNA binding sites, perhaps resulting from abnormal exchange of histones for transition proteins and/or protamines (Evenson, 1989; Evenson et al., 1991; Evenson and Jost, 1994). An increased number of cells with enhanced DNA staining characteristics, due to lack of appropriate sperm maturation, have often been reported in semen samples for certain subfertile patients (Evenson and Melamed, 1983; Spanò et al., 1984; Engh et al., 1992; Golan et al., 1997). The underlying assumption is that `hyperstainability' of human sperm chromatin can be interpreted as detrimental to delivery of the male genome in the oocyte. One of the advantages of SCSA is in the simultaneous detection of chromatin defects on single spermatozoa arising from the DNA and/or from the protamine level (Spanò et al., 1998). Additional SCSA advantages derive from the features of the FCM technique such as automation, standardization, objectivity, velocity, precision, reproducibility, and statistical robustness. In spite of the variety of different methods available to evaluate sperm chromatin maturity levels, the bulk of evidence points to the pivotal role of chromatin integrity during spermatogenesis and in the fertilization process. Lower packaging quality, found in morphologically normal spermatoza, may represent one of the major limiting factors in the fertilizing ability, thus stressing the importance of the tertiary and quaternary chromatin structure in the protection of genetic information and possibly in early post-fertilization events.
In the last decade, and in particular since the development of in-vitro fertilization techniques (IVF), the nuclear status of human sperm cells has shown to be a useful parameter in the assessment of male fertility. It has been shown that the probability of fertilization of oocytes in IVF is related to the frequency of spermatozoa with chromatin alterations (Hoshi et al., 1996; Lolis et al., 1996), and it has been reported that the vast majority of spermatozoa bound to human zona pellucida are characterized by normal chromatin packaging (Hoshi et al., 1996). It has been postulated that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation and, consequently, in fertilization failure (Sakkas et al., 1996; Samocha-Bone et al., 1998). Interestingly, sperm chromatin structure features do not seem to be related to conventional parameters of semen quality (Spanò et al., 1984; Evenson et al., 1991; Engh et al., 1992; Sakkas et al., 1995; Bianchi et al., 1996; Lolis et al., 1996; Fosså et al., 1997; Spanò et al., 1998). In particular, SCSA can identify the sperm subpopulations undergoing complete nuclear chromatin maturity (Evenson et al., 1989; Yossefi et al., 1994; Weissenberg et al., 1995; Golan et al., 1996) and can detect human sperm samples with suspected impaired fertility (Evenson et al., 1980, 1991; Evenson and Melamed, 1983; Evenson and Jost, 1994; Golan et al., 1996, 1997; Fosså et al., 1997).
Thus, changes in nuclear maturity can be used as an index of sperm quality in fresh semen and to check the quality of gametes after sperm manipulation. With regard to fresh semen, the establishment of normal values could be of great interest for their possible clinical application. At the current time, it is difficult to define what values are incompatible with normal human fertility since the interpretation of T parameters is still being explored and no data are available so far from studies specifically addressing the prognostic value of SCSA in predicting the human fertilizing potential. However, looking at the fraction of cells with abnormal chromatin in our cohort of normozoospermic subjects, we have observed a mean value for COMP T of 12.1 ± 9.8%. It is worthwhile noting that the reported upper limit value of 40.4% is associated with the semen of a donor showing the lowest sperm concentration (28x106/ml) and one of the highest fractions of sperm cell abnormalities (57%), still in the normal range of normozoospermia. Other studies support this observation; e.g. the mean value of the percentage of COMP T cells, evaluated in 18 Scandinavian normal donors was 9.4% and the upper limit of the normal range was 16% (Fosså et al., 1997), whereas in 45 American normal healthy men, COMP T was 16.8 ± 7.2% (Evenson et al., 1991). In another cohort of 277 Danish normal healthy individuals, we have found a mean value of 14.1% (Spanò et al., 1998). All these studies indicate that, in the general population, COMP T values are generally <20%. By and large, individuals with fertility problems characterized by low sperm concentration and motility values are often associated with a high fraction of malformed spermatozoa but also with anomalies in sperm chromatin packaging.
Furthermore, in this study, we have considered the swim-up technique which was used to select spermatozoa from both fresh and cryopreserved samples. We have demonstrated that swim-up techniques, which sorts a subpopulation of highly motile cells, select cells with better performances assessed by light and fluorescent microscopy, together with a remarkable improvement of the sperm chromatin features as evaluated by the SCSA. In particular, the fraction of COMP T markedly decreased in all samples and the mean value was almost one-third of the fraction observed in native semen samples. This subpopulation was also characterized by a lower X Green value (diminished access of AO due to the enhanced chromatin packaging) and lower SD Green, indicating that the variability of chromatin packaging in the whole sperm population was also reduced. Therefore, post-rise spermatozoa are characterized by superior and more homogeneous chromatin structure characteristics than that of unselected fresh (and, in the vast majority of cases, also cryopreserved) semen samples. Similar results were described in a recent paper by Molina et al. (1995); when swim-up was used to select highly motile cells, spermatozoa were produced which had improved nuclear maturity, i.e. reduced fluorescence intensity after propidium iodide uptake by FCM. The use of swim-up (and Percoll gradient centrifugation) methods was also shown to improve the percentage of spermatoza with normal chromatin structure in samples with poor initial quality (Golan et al., 1997).
The improvement of sperm chromatin quality in spermatozoa after swim-up contrasts with the deterioration observed in thawed samples after cryopreservation. This could be related to the variety of physical stresses the sample experiences during the cryopreservation also leading to a reduction of sperm motility and an increase of atypical sperm forms. A possible hypothesis is that cryopreservation, correctly carried out, is not able to damage spermatozoa per se, but can enhance defects already present in the sperm population. This has already been partially demonstrated using sperm function tests and CASA (Dondero et al., 1995). Recently, the FCM approach has also been used to monitor the deteriorating effects of cryopreservation on several sperm characteristics, such as mitochondrial function, membrane integrity, and cell viability (Kramer et al., 1993). Our study demonstrated that some of the SCSA-related parameters showed a general improvement over the subpopulation obtained by a physiological selection procedure, e.g. swim-up. This result is indicative of a strong correlation between `good' spermatozoa with a high probability of fertilizing and sperm chromatin integrity.
In conclusion, the SCSA provides a quantitative assessment of chromatin condensation in human semen samples. The sperm fraction with improved chromatin quality in a heterogeneous sample can be identified, since a strong correlation between motility and chromatin integrity may be an indication of fertilizing ability.
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Acknowledgments |
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Authors are deeply indebted to Doctors Pierluigi Altavista (ENEA, Rome) and Patrizio Pasqualetti (AFAR, Fatebenefratelli Hospital, Rome) for their help and expertise during the statistical analysis.
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3 To whom correspondence should be addressed
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References |
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Submitted on July 21, 1998; accepted on September 30, 1998.
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