Correlation between sperm motility and sperm chromatin structure assay parameters

Transcription

1 FERTILITY AND STERILITY VOL. 80, NO. 6, DECEMBER 2003 Copyright 2003 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. Correlation between sperm motility and sperm chromatin structure assay parameters Aleksander Giwercman, M.D., a Jonas Richthoff, M.D., a Henrik Hjøllund, Ph.D., b Jens Peter Bonde, M.D., b Katarina Jepson, a Birgitta Frohm, B.Sc., c and Marcello Spano, Ph.D. d Received January 8, 2003; revised and accepted April 28, Partially supported by a grant from the Italian Ministry of University and of Scientific and Technological Research on Biological Bases of Individual Susceptibility. Also supported by Swedish governmental funding for clinical research, the Swedish Cancer Society (grant no. 4423), the Swedish Research Council (grant no ), Gunnar Nilssons Cancerstiftelse, Crafoordska Stiftelse, Ove Tulefjords Fund, and the Foundation for Urological Research. Reprints requests: Aleksander Giwercman, M.D., Fertility Center, Malmö University Hospital, SE , Malmö, Sweden (FAX: ; aleksander. giwercman@kir.mas.lu.se). a Fertility Centre and Department of Urology, Scanian Andrology Center, Malmö University Hospital. b Department of Occupational Medicine, Aarhus University Hospital. c Department of Clinical Chemistry, Scanian Andrology Center, Malmö University Hospital. d Section of Toxicology and Biomedical Sciences, BIOTEC-MED, National Agency for New Technologies, Energy, and the Environment (ENEA) /03/$30.00 doi: /s (03) X Malmö University Hospital, Malmö, Sweden; National Agency for New Technologies, Energy, and the Environment (ENEA), Casaccia Research Center, Rome, Italy; and Aarhus University Hospital, Aarhus, Denmark Objective: To evaluate the association between chromatin structure and sperm motility. Design: Cross-sectional prospective study. Setting: Scanian Andrology Centre, Malmö, Sweden; ENEA Casaccia, Rome, Italy; and Department of Occupational Medicine, Aarhus University, Aarhus, Denmark. Patient(s): One hundred seventy-one males from Danish first pregnancy planner couples (group 1) and 278 Swedish military conscripts (group 2). Main Outcome Measure(s): Sperm chromatin structure assay (SCSA) parameters, DNA fragmentation index (DFI), high DNA stainable (HDS), and sperm motility, which was evaluated manually and by use of computer-aided sperm analysis (CASA). Result(s): A statistically significant negative correlation between DFI and the CASA percentage of motile sperms (group 1: r 0.53; group 2: r 0.38) was found. For the manual motility assessment, the correlation coefficients were slightly lower. Furthermore, HDS correlated negatively with CASA sperm motility (group 1: r 0.39; group 2: r 0.36) and percentage of World Health Organization category A motile sperm. In multiple linear regression analysis, concentration and SCSA parameters, but not the time of abstinence, were statistically significant predictors of sperm motility. Conclusion(s): There is a moderate correlation between sperm motility and SCSA parameters. The study supports the assumption that both SCSA and motility can be relatively independent predictors of male fertility. (Fertil Steril 2003;80: by American Society for Reproductive Medicine.) Key Words: Flow cytometry, sperm chromatin, sperm motility, CASA, SCSA Conventional semen analysis (1) consists of measuring a variety of semen parameters, including volume, ph, sperm concentration, vitality, and morphology, presence of leukocytes and immature germ cells, and sperm motility. This classical analysis is used to determine whether all or some of the parameters of an ejaculate fall within the range of those of normal fertile men. However, in at least 30% of cases of male factor infertility, the pathogenetic background is completely unknown. These cases are referred to as idiopathic infertility (2). It is believed that both diagnostic and prognostic power in andrology can be increased by increasing the information available from the conventional assessment and by the introduction of new semen quality biomarkers. Assessment of many of the conventional parameters is characterized by a relatively high level of intraand interlaboratory variation (3 7). The evaluation of sperm motility is especially influenced by subjective factors. However, the introduction of computer-aided sperm analysis (CASA) motility assessment turned out to be useful in improving prognostic power (8, 9). Methods focusing on the characterization of sperm chromatin condensation and stability are able to reveal hidden anomalies of the structural organization of sperm DNA and have been receiving growing attention. In particular, flow cytometric (FCM) approaches allow rapid measurements to be carried out with a high 1404

2 level of precision, objectivity, and repeatability and with sound results. Among the variety of FCM methods, the sperm chromatin structure assay (SCSA) (10, 11) is considered one of the most objective and robust measures of all tests in the infertility clinic. The SCSA defines abnormal chromatin structure as an increased susceptibility of sperm DNA to acid-induced denaturation in situ due to the occurrence of DNA breaks and/or derailments in protamine quantity and composition and/or an insufficient level of disulfide groups. The SCSA has, in a series of independent studies, been demonstrated to be a strong independent predictor of human fertility in vivo (12, 13) and in vitro (14): a threshold of 30% 40% of sperm with abnormal chromatin seems to be incompatible with term pregnancies, whatever the results from sperm concentration, motility, and morphology assessments. It is during the epididymal maturation that the sperm acquires the capacity for progressive motility. The DNA is reorganized around protamine molecules during testicular spermiogenesis (elongating and elongated spermatid stage). In the epididymis, the cysteine-thiol groups are oxidized to provide the highly rigid structure (15). It is speculated that the generation of a more protected genome (sperm has practically no DNA repair system) and of a more hydrodynamic sperm head can speed transit through the female reproductive tract. Therefore, both processes, namely, nucleus remodeling and acquisition of motility, seem essential to fulfill the primary goals of the male gamete genome, that is, a successful fecundation and a fully sustained pregnancy. Consequently, one can speculate that any impairment to these concurrent, apparently noncorrelated, differentiation pathways should hamper the proper expression of the sperm reproductive capability. Based on this, one interesting question is to determine the level of independence or correlation between motility and chromatin integrity. Conflicting results have been obtained in studies attempting to correlate these two features of the human sperm, both of which are necessary to fertilize. Generally, in performing the SCSA, a statistically significant but quite low level of correlation between SCSA and World Health Organization (WHO) parameters has been found. A relative independence of SCSA parameter values from sperm concentration and morphology is reported in all the studies published so far (4, 12, 16). Furthermore, SCSA parameters have been reported to be relatively independent of the motility status of the spermatozoa, although the levels of the correlation were quite variable, ranging from nonsignificant (14) up to 0.53 (17, 18). However, the available studies were generally based on a relatively small population and/or selected groups of individuals, mainly patients recruited from infertility clinics, who usually showed a broad range of pathogeneses that influenced their reproductive function. Researching the association between the two sperm characteristics both known to be predictors of male fertility potential is important to improve our understanding of the mechanisms involved in the impairment of male reproductive functions and the development of new, more closely targeted treatment strategies. In this study, sperm motility, as objectively evaluated by CASA, has been determined as a function of sperm chromatin stability, as objectively evaluated by the FCM SCSA. Two SCSA parameters have been considered, namely, the DNA fragmentation index (DFI, formerly known as COMP T) and the high DNA stainable (HDS, formerly known as high green or HGRN) fraction (11) and have been correlated with the percentage of progressively motile sperm in a population of 171 Danish male first pregnancy planners and 278 Swedish military conscripts who represent the general population of males of different ages. MATERIALS AND METHODS Our study populations were recruited through two separate surveys of reproductive functions. The first study originally included 430 Danish first pregnancy planners, and the other study included 305 military conscripts from southern Sweden. Both studies were approved by local ethics committees, and the men were included after giving written informed consent. The details of the two studies are described elsewhere (8, 13, 19). Group 1: First Pregnancy Planners During the period , 430 Danish first pregnancy planning couples were enrolled through two centers, in Copenhagen and in Aarhus. In 171 of the 231 cases, SCSA as well as CASA analysis was performed. The remaining 60 individuals were excluded because of azoospermia, insufficient material left for SCSA analysis, lack of CASA measurements in the pilot part of the First Pregnancy Planner Study, or omission of SCSA analysis in men who did not have intercourse with their partner between day 11 and 20 of the menstrual cycle. These details are given in previous reports on this study (9, 13). The men were aged years (mean, 28.4 years), and none of them had ever attempted to achieve a pregnancy or had involuntarily achieved a pregnancy. Thus, these individuals are considered to belong to the general population of males attempting to conceive their first child. Sperm chromatin analysis was performed on the sample delivered by each male at the time he entered the study. Group 2: Military Conscripts Approximately 95% of all Swedish males undergo a medical health examination before military service. Only those with serious chronic diseases are a priori excluded. Therefore, the group of conscripts closely reflects the general population of young Swedish males. A total of 2,255 men born during the period and living within an area of 60 km from the city of Malmö in southern Sweden were asked to participate. A total of 305 (13.5%) were accepted FERTILITY & STERILITY 1405

3 for entrance into the study and signed a written consent form. From 278 of them, enough material was available to perform CASA and SCSA analysis. The men were aged years (mean, 18.2 years). Semen Analysis The men included in both groups delivered a semen sample by masturbation into a wide-mouth plastic cup at home (group 1) or in the laboratory (groups 1 and 2). The sample was available for analysis within 60 minutes after ejaculation. The men in group 2 were asked to abstain from sex for at least 48 hours before sample delivery, and the length of the actual abstinence period was recorded in both groups. Sperm concentration was analyzed largely according to WHO 1992 and 1999 recommendations (1, 20). The only exception was the use of a Makler chamber for analysis of samples from group 2, whereas an improved Neubauer hemocytometer was used for analysis of samples from group 2. Also, the manual analysis of motility was done as recommended by WHO (1, 20). Spermatozoa were characterized as rapidly progressively motile (A), slowly progressively motile (B), nonprogressively motile (C), or immotile (D). In addition to the manual analysis, the motility was assessed by CASA (see below). After 30 minutes of liquefaction, 0.2 ml aliquots of neat semen were coded and frozen in cryotubes at 80 C for later FCM SCSA analysis (see below). CASA Analysis The percentage of motile, locally motile, and immotile sperms was assessed by use of the CRISMAS (Image House, Copenhagen, Denmark) CASA system, as described elsewhere (9). In brief, the analysis was performed in a 10- m Makler chamber at 37 C. The samples were, if necessary, diluted in a phosphate buffer. The motility assessment was based on capture sequences of 64 images (25 MHz), and for each sample at least 100 sperms were analyzed. The designation of the motility status was based on the level of the curvelinear velocity (VCL), with a VCL 25 m/second for motile sperm, 5 25 m/second for locally motile sperm, and 5 m/second for immotile sperm. Identical equipment and conditions were used for analysis of both samples. FCM SCSA Samples from group 1 were analyzed in Rome, Italy, and from group 2 in Malmö, Sweden. In both laboratories, identical procedures were used (see below). However, to ensure comparability of results, a quality control exercise based on 181 samples from group 2 (from which enough material was available) was performed blindly in both the Malmö and Rome flow cytometry facilities. The SCSA was done according to the procedure described elsewhere (11, 13). On the day of analysis, the samples were quickly thawed in a 37 C water bath and used immediately. A total of cells were treated with a low-ph (1.2) detergent solution containing 0.1% Triton X-100, 0.15 M NaCl, and 0.08 N HCl for 30 seconds and then stained with 6 mg/l of purified acridine orange (AO; Molecular Probes, Eugene, OR) in a phosphate-citrate buffer, ph 6.0. Cells were analyzed by a FACSort in Malmö and by a FACScan in Rome (both flow cytometers are manufactured by Becton Dickinson, San Josè, CA) equipped with an air-cooled Argon (Ar) ion laser. For the flow cytometer setup and calibration, aliquots were used from a normal human ejaculate sample retrieved from our laboratory repository. Samples were measured blindly, and the code was broken only at the end of the measurement series. Five thousand events were accumulated for each measurement. Samples were measured twice, and the results provided refer to the mean value of the two FCM measurements. Under these experimental conditions, when excited with a 488-nm light source, AO intercalated with double-stranded DNA emits green fluorescence and AO associated with single-stranded DNA emits red fluorescence. Thus, sperm chromatin damage can be quantified by the FCM measurements of the metachromatic shift from green (native, doublestranded DNA) to red (denatured, single-stranded DNA) fluorescence and displayed as red (fragmented DNA) vs. green (DNA stainability) fluorescence intensity cytogram patterns. The guidelines described in a recent publication (11) have been adopted, and, therefore, we have expressed the extent of DNA denaturation in terms of DFI (formerly, the T function), which is the ratio of red to total (red plus green) fluorescence intensity, by using the ListView software (Phoenix Flow Systems, San Diego, CA). This conversion is necessary to correctly calculate the percentage of spermatozoa with a nondetectable DFI (formerly, the main or normal population of cells) and a detectable (moderate and high) DFI (formerly collectively termed COMP T, or cells outside the main population). The DFI value was calculated for each sperm cell in a sample, and the detectable DFI values, calculated on the DFI frequency histogram, were also used to compare our results with the COMP T values reported in the existing literature. Additionally, we have also considered the fraction of HDS cells (formerly, the HGRN fraction). The percentage of HDS cells was calculated by setting an appropriate region on the scattergram (abscissa: red fluorescence, fragmented DNA; ordinate: green fluorescence, native DNA stainability) and considering as immature those spermatozoa that exhibit a green fluorescence intensity higher than the upper border of the main cluster, which represents the sperm population with nondetectable DFI. Consequently, we will offer the results relative to the proportion of spermatozoa with increased levels of fragmented DNA and to the proportion of immature spermatozoa because both parameters are currently being considered in infertility investigations (11) Giwercman et al. Chromatin structure and sperm motility Vol. 80, No. 6, December 2003

4 TABLE 1 Background characteristics considering abstinence duration and sperm parameters in the two populations of males included in the present study. Group 1, first pregnancy planners (n 171) Group 2, military conscripts (n 278) Mean (SD) Median Range Mean (SD) Median Range Duration of abstinence (hours) 110 (99) (54) Sperm concentration (10 6 /ml) 70 (57) (65) DNA fragmentation index (%) 10 (8.6) (9.0) High DNA stainable (%) 17 (9.9) (4.9) Motility manually assessed (%) A 46 (17) (17) B C 16 (5.8) (120) D 38 (16) (13) Motility assessed by computer-aided sperm analysis (%) Motile 49 (21) (22) Locally motile 44 (16) (16) Immotile 7.2 (7.0) (23) Statistical Analysis Statistical analysis was performed using the SPSS 11.0 software (SPSS Inc., Chicago, IL). Logarithmic transformation of DFI, HDS, time of abstinence, and sperm concentration was undertaken to obtain normal distribution of the data. Bivariate associations between SCSA parameters and abstinence time as well as sperm concentration and motility characteristics were evaluated by Pearson s correlation coefficient. Percentages for motility groups B and C were added into a joint category since this movement s characteristics correspond to the subgroup of locally motile as determined by CASA. Bivariate correlation analysis was also applied for comparison of DFI results, which were obtained on 181 samples from group 2 and measured in the two laboratories performing SCSA. Finally, the effects of SCSA parameters, sperm concentration, and the time of abstinence on percentage progressively motile sperms (manual analysis) as well as on percentage of motile cells (CASA analysis) were evaluated by use of a linear multiple regression model. RESULTS The background characteristics of the two groups of males are given in Table 1. Analysis of the samples from the two groups gave similar results. By use of bivariate analyses, a statistically significant positive correlation was found between the DFI and length of the abstinence period as well as the percentage of immotile sperm as assessed manually and by means of CASA. Statistically significant negative correlations were seen between DFI and sperm concentration as well as between the percentage of progressively motile sperms and CASA motile cells. Pearson s correlation coefficients between the DFI and the percentage of motile sperms varied between 0.33 and 0.43 for the manual assessment and between 0.38 and 0.53 for CASA motile sperm values (Fig. 1). Similar correlations were found for HDS and abstinence period length as well as for sperm parameters. The results obtained in the two groups of subjects were almost identical except that in group 1, but not in group 2, there was a statistically significant positive correlation between the percentage of CASA locally motile sperm and HDS. These results are summarized in Table 2. The bivariate correlation coefficients between the independent variables included in the multivariate analysis were 0.5 or less, which, therefore, were considered as grossly noncollinear. In group 2, the multivariate analysis did show that sperm concentration and DFI were the strongest and equal predictors of sperm motility assessed manually as well as by means of CASA. HDS was somewhat weaker but was statistically significantly associated with motility parameters, whereas the association with the length of the period of abstinence was no longer statistically significant. The R 2 values were 0.3 for CASA motile sperms and 0.16 for manually assessed motility. The results are shown in Table 3. The analysis performed on pregnancy planners data gave similar results (data not shown). A comparison of the DFI measurements performed in two laboratories revealed a high level of correlation (r 0.90; P.0005). The mean ratio between the Rome and Malmö values was 1.01 (SD 1.05) (Fig. 2). FERTILITY & STERILITY 1407

5 FIGURE 1 Scatterplot illustrating the association between DFI and CASA percentage motile sperms in 278 military conscripts from Sweden. Pearson s r DISCUSSION Based on two populations representing two age categories of subjects from the general male population, we found a statistically significant moderate level of association between the SCSA parameters DFI and HDS and parameters of sperm motility as evaluated manually and by use of CASA. TABLE 2 Bivariate Pearson s correlation coefficient between sperm chromatin structure assay parameters DNA fragmentation index and high DNA stainable and time of abstinence, sperm concentration, and sperm motility parameters in the cohorts of military conscripts and in first pregnancy planners. Group 1, first pregnancy planners (n 171) Group 2, military conscripts (n 278) DFI HDS DFI HDS r P r P r P r P Duration of abstinence (hours) Log sperm concentration Motility manually assessed (%) A B C D Motility assessed by computer-aided sperm analysis (%) Motile Locally motile Immotile Giwercman et al. Chromatin structure and sperm motility Vol. 80, No. 6, December 2003

6 TABLE 3 Effects of sperm chromatin structure assay parameters on manually and computer-aided sperm analysis (CASA) assessed percentage of motile sperms in 278 military conscripts obtained by multivariate regression analysis. Sperm concentration and abstinence time were included as the other independent variables. % Rapid progressively motile (manual analysis) % Motile (CASA analysis) (Nonadjusted) (Adjusted) P (Nonadjusted) (Adjusted) P Intercept DNA fragmentation index High DNA stainable For CASA percentage motile sperms, the fact that the correlation coefficients were in the range of indicated that less than 30% of the variation in sperm motility characteristics could be explained by factors related to sperm chromatin integrity as evaluated by SCSA. Sperm motility was assessed by use of the standard manual procedure as well as by CASA. Sperm motility scoring is subject to significant inter- and intraobserver variation (3, 5, 6), which can be reduced by the use of CASA systems (21). This might be the cause of slightly higher correlation coefficients for associations between SCSA and motility parameters when the latter were measured by means of CASA. FIGURE 2 Bivariate correlation between DFI measurements on 181 ejaculates from military conscripts, performed in Malmö and in Rome. Pearson s r FERTILITY & STERILITY 1409

7 TABLE 4 Previous studies on correlation between sperm chromatin structure assay DNA fragmentation index and percentage of motile sperm. Subjects n Correlation coefficient (r) Reference General population (4) Infertility patients (12) Infertility patients (45) Infertility patients 24 NS (14) Infertility patients (47) Infertility patients (48) Infertility patients (17) Infertility patients (18) Cancer patients 37 NS (49) This is, to our knowledge, the first study of a significant number of men from the general population that shows a correlation between sperm chromatin characteristics and motility. Previous studies have indicated such a relationship but were based on a limited number of individuals and with one exception (4) included men recruited from infertility clinics or from oncology departments (Table 4). Men referred for infertility problems comprise from a pathogenetic point of view a heterogeneous group (22). Aberrations in sperm motility, often seen in this group of patients, might be due to a multitude of testicular and post-testicular events acting as potential confounders when looking for an association between sperm chromatin and the function of spermatozoa. Classical sperm parameters, including motility, were previously shown to be closely interrelated with each other (8). Similarly, an association between SCSA parameters and sperm concentration as well as the length of the abstinence period has been reported (16, 23). We have, therefore, performed a multivariate regression analysis including not only sperm concentration and the duration of abstinence, but also the SCSA measures of DFI and HDS. This analysis confirmed that sperm chromatin parameters are associated with sperm motility independent of the other parameters. The moderate association between DFI and motility indicates that normally motile sperm may or may not have an intact DNA molecule. The SCSA parameter DFI is believed to reflect mainly the population of sperm with DNA breaks, and it has been demonstrated to be strongly associated with the results of other methods developed to measure the fraction of sperm with DNA breaks, namely, the Comet and the TUNEL assays (17, 24 26). Actually, the SCSA results are in line with the results obtained after TUNEL assay to detect spermatozoa with DNA breaks whose association with motility ranges from poor to moderate (17, 27 29). However, in a recent survey carried out on almost 100 individuals, TUNEL positivity did not correlate with sperm motility (30). It should be remembered that these data have been obtained from subjects with fertility problems. It is noteworthy that there is also evidence that sperm with fragmented DNA as evaluated by the TUNEL assay can exhibit ultrastructural features indicating impaired motility (31). Furthermore, in a recent study of 60 fertility clinic patients in whom sperm DNA damage was assessed by single-cell electrophoresis (Comet assay), the parameter tail length (of the comets), which mirrors the level of DNA breaks, was statistically significantly, although poorly, associated with motility (r 0.29) (32). Besides the association with DFI, a marker of sperm with fragmented DNA, motility was also correlated to the size of the HDS fraction, which is believed to be directly related to sperm with defects in the correct protamination process. It has been reported that sperm maturation defects resulting in an incomplete replacement of nuclear histones might be associated with human infertility (33). Subsequent studies have reported that some infertile men have a diminished level of protamine 2 in their sperm and that the low level of P2 may be due to incomplete processing of protamine 2 precursors (34, 35). It looks like the variations in expression observed in infertility patients may be due to uncoupling of the expression of protamine 1 and protamine 2 genes, which is exacerbated by post-translational cleavage events that may be responsible for the variations of protamine expressions observed. In fact, it has recently been reported that abnormal sperm protamine levels are a common defect in infertility patients but not in donors with known fertility (36) and patients with no protamine 2 at all had significantly fewer morphologically normal forms and, interestingly, less progressively motile sperm. However, the way one looks at the protamination level could be of importance. For example, using the fluorescence chromomycin A3 (CMA3) method, which can detect sperm with an insufficient level of chromatin protamination, there was no significant change in CMA3 staining after swim-up preparation, but a significant decrease in CMA3 staining was reported after density gradient centrifugation (37). We can speculate that sperm histone replacement by protamines may facilitate improved sperm motility in addition to protecting sperm DNA from damage and may reset or imprint the male genome. It should be taken into account that during epididymal transit sperm chromatin is always further stabilized by the progressive oxidation of protamine-free thiol groups (38), which can be mirrored by the progressive decrease of AO binding to DNA as evaluated by the FCM SCSA (39). Therefore, besides abnormal deposition of sperm protamines during spermiogenesis, incomplete oxidation of sperm protamine thiol groups during epididymal transit can also lead to enhanced susceptibility of sperm DNA denaturation (40). A recent study (18) showed that, in a group of 76 men, sperm thiol content was correlated positively with sperm chromatin integrity as evaluated by SCSA (r 0.53, P.0001). In the 1410 Giwercman et al. Chromatin structure and sperm motility Vol. 80, No. 6, December 2003

8 same study, a significant negative relationship between the sperm thiol content and motility was found (r 0.65, P.0001). In our study, the association between the HDS fraction and motility can also be explained by the fact that this fraction could represent highly DNA-stainable cells because of a lower disulfide content (41). Interestingly, in knockout mice deficient for the expression of transition proteins, which are believed to be important for histone displacement and chromatin condensation during mammalian spermatogenesis, the epididymal sperm phenotype showed, along with chromatin structure abnormalities (also assayed by the SCSA), a strong reduction in sperm motility and an increase in abnormal tail morphology (42, 43). The possible link between sperm chromatin integrity and motility has also been indirectly detected, and other studies have offered clues that these prominent sperm features could be somehow associated. For example, when checking gamete quality before and after separation in the procedures used to select the populations of spermatozoa with the highest chances to fertilize (Percoll, swim-up, etc.) and measuring sperm chromatin integrity by the SCSA, in many instances, the sperm subpopulations obtained, besides being enriched with sperm with the best morphology and, above all, motility, were also characterized by a net decrease in the DFI values (44 47). This supports the pragmatic hypothesis about the importance of having the best motility performances together with the highest level of sperm with intact chromatin to increase the chance of a successful fertilization. Our results reinforce the finding that sperm motility and chromatin integrity are significantly associated, and we offer a measure of this relationship, which, at any rate, remains only moderate. Spermatozoa with excellent chromatin structure but without motility or with other abnormalities will still be infertile. However, motile and otherwise normal spermatozoa with damaged DNA and chromatin can certainly have an impaired function. Therefore, it is suggested that both parameters, being strong and independent predictors of the fertility status when evaluated by operator-independent, objective methods such as FCM and CASA, can complement each other, which provides additional diagnostic and prognostic criteria for male factor infertility evaluations. This is also of importance for clinical applications from assisted reproduction techniques because of the high probability of using spermatozoa with broken DNA and/or chromatin abnormalities, which may potentially lead to the transmission of defective genetic material into the female egg and, consequently, to possible abnormal embryo development. Acknowledgments: The authors thank the Danish First Pregnancy Planners Study Team. The Danish First Pregnancy Planner Study is a collaborative follow-up study on environmental and biological determinants of fertility. The project is coordinated by the Steno Institute of Public Health, Aarhus University, and is undertaken in collaboration with the Department of Growth and Reproduction, National University Hospital, Copenhagen. The team includes Jens Peter E. Bonde, Niels Henrik I. Hjøllund, Tina Kold Jensen, Tine Brink Henriksen, Henrik A. Kolstad, Erik Ernst, Anna-Maria Andersson, Aleksander Giwercman, Niels Erik Skakkebæk, and Jørn Olsen. References 1. World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical mucus interactions. 4th ed. Cambridge: Cambridge University Press, Nieschlag E. Scope and goal of andrology. In: Nieschlag E, Behre HM, eds. Andrology: male reproductive health and dysfunction. Berlin: Springer, 2000: Neuwinger J, Behre HM, Nieschlag E. 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