TECHNIQUES AND INSTRUMENTATION

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1 FERTILITY AND STERILITY VOL. 82, NO. 6, DECEMBER 2004 Copyright 2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A. TECHNIQUES AND INSTRUMENTATION Relationship between human semen parameters and deoxyribonucleic acid damage assessed by the neutral comet assay Ana T. Trisini, B.S., a Narendra P. Singh, M.B.B.S., M.S., b Susan M. Duty, M.S.N., Sc.D., a,c and Russ Hauser, M.D., Sc.D., M.P.H. a,d Harvard School of Public Health, and Massachusetts General Hospital, Boston, Massachusetts Received January 23, 2004; revised and accepted May 5, Supported by grants ES09718 and ES00002 from the National Institute of Environmental Health Sciences, National Institutes of Health. Reprint requests: Russ Hauser, M.D., S.c.D., M.P.H., Harvard School of Public Health, Department of Environmental Health, Occupational Health Program, Building 1, Room 1405, 665 Huntington Avenue, Boston, Massachusetts (FAX: ; rhauser@hohp.harvard.edu). a Department of Environmental Health, Occupational Health Program, Harvard School of Public Health. b Department of Bioengineering, University of Washington, Seattle, Washington. c School for Health Studies, Nursing Programs, Simmons College, Boston, Massachusetts. d Vincent Memorial Obstetrics and Gynecology Service, Andrology Laboratory and In Vitro Fertilization Unit, Massachusetts General Hospital /04/$30.00 doi: /j.fertnstert Objective: To explore the association between semen parameters and sperm DNA damage. Design: Cross-sectional. Setting: Andrology clinic. Patient(s): Two hundred fifty-seven men undergoing infertility assessment. Intervention(s): None. Main Outcome Measure(s): Sperm concentration and motility were measured using computer-assisted sperm analysis; morphology was scored using the strict criteria. The neutral comet assay was used to measure sperm DNA damage. Comet assay parameters included comet extent, percent DNA in the comet tail, and tail distributed moment, an integrated measure of length and intensity. We also scored cells that were too long to measure ( 300 m), which we referred to as cells with high DNA damage. Result(s): Men older than 35 years had a statistically significant increase in the number of cells with high DNA damage as compared with younger men. In age-adjusted regression analyses, the most consistent associations were found between semen parameters and the number of cells with high DNA damage. For an interquartile range change in the number of cells with high DNA damage, sperm concentration declined /ml, motility declined 4.3%, and morphology declined 0.5%. Comet extent and percent DNA in the comet tail were also associated with a decline in sperm concentration and motility, respectively. Conclusion(s): Although there were associations between semen and comet assay parameters, their magnitudes were weak, suggesting that the comet assay provides additional independent information on sperm function. (Fertil Steril 2004;82: by American Society for Reproductive Medicine.) Key Words: Comet assay, DNA integrity, microgel electrophoresis, sperm There is ongoing research in developing new methods to complement conventional semen analyses routinely used to assess infertility. One endpoint of interest is sperm DNA integrity, which can be assessed by a variety of assays, such as sperm chromatin structure assay (SCSA) (1), terminal deoxynucleotidyl transferase-mediated dudp nick-end labeling (TUNEL) assay (2), in situ nick translation (ISNT) (3), and single cell microgel electrophoresis (comet) assay, neutral and alkaline (4, 5). Before incorporating these methods into the clinical setting, further research is needed to determine the relationship between semen parameters and sperm DNA integrity, as well as to determine whether sperm DNA integrity measures are predictive of fertility endpoints (6). A number of studies have investigated the relationship between human sperm DNA damage and semen parameters, that is, concentration, morphology, and motility (7 14). In several different study populations, using different assays to measure DNA damage, investigators found associations between some semen parameters and sperm DNA integrity (7, 10, 12, 14), whereas several others did not find associations (8, 9). Irvine et al. (10) evaluated the association between semen parameters and DNA integrity among a group of infertile patients and a group of normozoospermic donors using three techniques: alkaline comet assay, 1623

2 ISNT condensed, and ISNT decondensed. They showed that semen parameters, especially sperm concentration, were inversely correlated with the comet assay parameters, ISNT decondensed, and ISNT condensed. The comet assay had the highest resolution for DNA fragmentation in that it allowed separation of the groups of individuals. Potential mechanisms of sperm DNA damage include [1] defective chromatin condensation during spermiogenesis (15), [2] initiation of apoptosis during spermatogenesis or during transport of sperm through male or female genital tracts (16), and [3] oxidative stress by reactive oxygen species produced internally or externally (17, 18). These three mechanisms might independently or codependently be responsible for sperm DNA damage. The first mechanism postulates that sperm does not mature completely during spermiogenesis and that DNA breaks are therefore unable to physiologically re-ligate when chromatin rearrangement occurs during the replacement of histones by protamines (15). Furthermore, many investigators have correlated the presence of DNA damage with poor chromatin packaging (19 21). The second hypothesized mechanism relates to apoptotic DNA cleavage in germ cells (16). Sperm DNA damage might result from an abortive apoptosis, which occurs in the later stages of germ cell development, resulting in dying sperm cells not being properly eliminated by the testis. Finally, a third hypothesized mechanism attributes sperm DNA damage to excessive reactive oxygen species production in testicular and posttesticular sites (22 24). The three mechanisms described also might contribute to alterations in semen parameters. For instance, there is evidence that oxidative DNA damage, measured as 8-OH-dG in sperm DNA, inversely correlates with sperm concentration (25) as well as with sperm motility and morphology (26). In both of these studies, investigators used high-performance liquid chromatography with electrochemical detection to demonstrate associations between oxidative DNA damage in sperm and semen parameters. There is also evidence suggesting that apoptosis plays a role in semen quality. In a study of 102 infertility clinic patients, inverse correlations were found between phosphatidylserine expression with additional labeling from the vital dye propidium iodide (Annexin V/PI binding assay) and sperm concentration and motility (27). There was also an inverse correlation between DNA double-strand breaks (TUNEL assay positive cells) and sperm concentration. The Annexin V/PI assay measures apoptosis during an early stage of the process, whereas the TUNEL assay measures apoptosis at a relatively late stage of the process. Although Ricci et al. (28) did not find an overall association between apoptotic index and semen quality when they classified semen samples according to World Health Organization (WHO) guidelines, there was an inverse correlation between apoptotic index and sperm concentration and morphology in men with normal semen parameters. However, apoptotic index did not correlate with semen parameters in men with abnormal semen parameters. Because similar mechanisms might be responsible for sperm DNA damage and semen quality, the present study was designed to explore the association between semen parameters and neutral comet assay parameters in a large cohort of men attending the Massachusetts General Hospital Andrology Clinic in Boston, Massachusetts. We also explored the relationship between comet assay parameters and age and smoking status. MATERIALS AND METHODS Patients The study was approved by the human subject committee institutional review boards of the Harvard School of Public Health and Massachusetts General Hospital. All authors declared no conflict of interest in connection with this study or its contents. All participants gave written, informed consent. The study s participants were the male partners in couples who attended the Vincent Burnham Andrology Laboratory at Massachusetts General Hospital between January 2000 and April 2002 for treatment for inability to achieve a pregnancy. Sixty-six percent of eligible men, between 20 and 54 years of age, agreed to participate and provided written, informed consent. Men presenting for postvasectomy semen analysis and men receiving treatments for infertility, such as hormonal treatments, were excluded. Height and weight were measured, and a questionnaire was used to collect information on medical history and lifestyle factors. Semen Analyses Each man produced a single semen sample by masturbation into a sterile plastic specimen cup. The sample was liquefied at 37 C for 20 minutes before analysis. The men were instructed to abstain from ejaculation for 48 hours before producing the semen sample and to complete a questionnaire on the length of the sexual abstinence period. All semen samples were analyzed for sperm concentration and motion parameters by computer-aided semen analysis (CASA; Hamilton-Thorn Version 10HTM-IVOS, Beverly, MA). The setting parameters and the definition of measured sperm motion parameters for CASA were established by the manufacturer. To measure both sperm concentration and motility, aliquots of semen samples (5 L) were placed in a prewarmed (37 C) Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). A minimum of 200 sperm cells from at least four different fields was analyzed from each specimen. The percentage of motile sperm was defined as WHO grade A sperm (rapidly progressive with a velocity 25 m/s at 37 C) plus B grade sperm (slow/ sluggish progressive with a velocity 5 m/s but 25 m/s) Trisini et al. Semen and comet assay parameters Vol. 82, No. 6, December 2004

3 Seven CASA sperm motion parameters were measured, and as expected many were strongly correlated with each other because they describe different aspects of the same movement. Measures of progression average path velocity (VAP) and straight-line velocity (VSL) were highly correlated (r 0.95, P.0001), indicating they were likely measuring a similar characteristic of sperm movement. As a measure of progression, VSL was chosen over VAP because it is a direct measurement as opposed to a mathematically smoothed value. Curvilinear velocity (VCL) was chosen as a measure of vigor. The chosen measure of swimming pattern was LIN ( VSL/VCL 100) because VSL and VCL are components of LIN. Therefore, measures of progression (VSL), vigor (VCL), and swimming pattern (LIN) were chosen for in-depth statistical analyses. These three measures are also not as heavily dependent on the type of CASA instrument used, allowing for some comparison with results from other studies. Using the feathering method from the 1999 WHO manual (29), at least two slides were made for each fresh semen sample. The resulting thin smear was allowed to air dry for 1 hour before it was stained with a Diff-Quik staining kit (Dade Behring, Düdingen, Switzerland). Spermatozoa were scored as normal or abnormal using the strict criteria by Kruger et al. (30). A minimum of 200 sperm cells was scored from two slides for each specimen. Results were expressed as the percentage of normal spermatozoa. All slides were prepared and scored by the same person. Comet Assay We previously published data from our laboratory on the reliability of the comet assay in cryopreserved human sperm (31). We demonstrated a dose response relationship between comet assay parameters and x-ray dose. The entire comet assay procedure is conducted under low, indirect, incandescent light (60 W) to minimize lightinduced damage to sperm DNA. After thawing, semen (approximately sperm cells) was mixed with 0.7% agarose (3:1 high resolution; Amresco, Solon, OH) and embedded between two additional layers of agarose on specially designed glass slides (Erie Scientific, Portsmouth, NH). The slides were transferred to two different solutions for enzyme treatment for specific time periods, crucial steps for decondensing sperm chromatin and allowing migration of broken DNA out of the nucleus. Slides were electrophoresed under neutral conditions at 12 V and 130 ma for 1 hour at room temperature, followed by precipitation of the DNA and its fixation in microgels. After the slides were air-dried and stained, fluorescent comet patterns were examined under 400 magnification with a Leica fluorescence microscope model DMLB (Leica, Wetzlar, Postfach, Germany), equipped with fluoroisothiocyanate filter combination (Leica, Wetzlar), excitation at 490 nm, dichroic 500 nm, emission 510 nm. Image Analysis VisComet image analysis software, kindly donated by Impuls Bildanalyse GMBH (Gilching, Germany), was used to measure comet extent, percent DNA in tail (% DNA in tail), and tail distributed moment (TDM) on 100 sperm cells in each semen sample. Comet extent is a measure of total comet length from the beginning of the head to the last visible pixel in the tail; this measurement is similar to that obtained by manual analysis using an eyepiece micrometer. The % DNA in tail is a measurement of the proportion of the total DNA that is present in the tail. The TDM is an integrated value that takes into account both the distance and intensity of comet fragments; the formula used to calculate TDM is: Where M dist (I * X) I I sum of all intensity values that belong to the head, body, or tail. X x-position of intensity value. In addition to these three comet assay parameters, cells with high DNA damage (CHD), those too long to measure with VisComet ( 300 m), were tallied and used as a fourth measure of DNA damage. Traditionally, in the neutral comet assay, CHD are not scored. Because of the presence of CHD in most of the study participants, more than 100 cells might have been screened and scored to allow for the measurement of comet extent, % DNA in tail, and TDM in 100 cells per patient. Statistical Analyses For data analysis, Statistical Analysis Software (SAS), version 8.2 (SAS Institute, Cary, NC), was used. Descriptive analyses of patient characteristics were performed. In univariate and multiple regression analyses, the mean comet extent, % DNA in tail, and TDM of 100 cells per person was used as the independent variable. Because mean comet extent and TDM were normally distributed, they were not transformed in the regression analyses. However, % DNA in tail and CHD were not normally distributed; therefore, analyses using both untransformed and log or arcsine transformations, respectively, were performed (32). Because the results and their interpretation did not vary, we chose to present only the untransformed % DNA in tail and CHD results for ease of interpretation. The regression coefficients are expressed as the change in the semen parameter per unit change in the comet assay parameter. All regression analyses were adjusted for age and abstinence time; however, because abstinence time did not alter the results, it was not retained in the regression models. Spearman correlation coefficients were used to determine correlations among semen parameters and comet parameters. FERTILITY & STERILITY 1625

4 TABLE 1 Subject demographics, and semen and comet parameters (n 257). Wilcoxon signed-rank tests were used to compare comet and semen analysis parameters by categorical variables, such as smoking status (current, ex-smoker, never smoker) and age ( 35 years vs. 35 years). RESULTS n (%) Mean (SD) (5.5) Race b White 209 (82.3) Black/African American 15 (5.9) Hispanic 14 (5.5) Other 16 (6.3) Smoking status c Never smoker 183 (73.2) Ever Smoker 67 (26.8) Current smoker 22 (8.8) Ex-smoker 45 (18.0) Semen parameters d Sperm concentration (sperm 10 6 /ml) (95.5) Patients sperm/ml 36 (14.0) Sperm motility (% motile sperm) 51.5 (23.4) Patients 50% motile sperm 109 (42.4) Sperm morphology (% normal sperm) 7.0 (4.3) Patients 4% normal morphology 58 (22.7) VCL ( m/s) 78.0 (18.7) VSL ( m/s) 45.4 (10.8) LIN (%) 58.2 (9.2) Note: n number of patients; SD standard deviation; VCL curvilinear velocity; VSL straight-line velocity; LIN linearity. a Age missing for four patients. b Race missing for three patients. c Smoking information missing for seven patients. d VCL, VSL, and LIN missing for one patient; Kruger strict criteria used for morphology determination. The study population consisted of 257 men. Demographic and semen analysis distributions are described in Table 1. The mean ( SD) age was 36.1 ( 5.5) years old, and ranged from 22 to 54 years. Race was self-reported as Caucasian (82.3%), Black/African American (5.9%), Hispanic (5.5%), and Other (6.3%). Most of the men were never smokers (73%), 9% were current smokers, and 18% were ex-smokers. Thirty-six men (14.0%) had a sperm concentration of 20 million/ml, 109 men (42.4%) had 50% motility, and 58 men (22.7%) had 4% normal morphology. These belowreference values were not mutually exclusive; therefore, study patients might have contributed to more than one group. The mean ( SD) VCL, VSL, and LIN were 78.0 ( 18.7) m/s, 45.4 ( 10.8) m/s, and 58.2% ( 9.2), respectively. The distribution of four comet assay parameters is shown in Table 2. There were fourfold to 10-fold ranges in the distributions. Comet extent ranged from 53.4 to m, with a median of m; % DNA in tail ranged from 7.0% to 64.4%, with a median of 21.5%; and TDM ranged from 28.6 to m, with a median of 58.4 m. The CHD ranged from 0 to 95 cells and had a median of seven cells. In Figure 1, panels A to C demonstrate the heterogeneity of comet tail lengths within an individual, and panel D depicts the comet cell referred to as a CHD. On the basis of the age distribution in our patients, we set a cut point age of 35 years to categorize men as old ; men aged 35 years were categorized as young. In older men, there was a statistically significant increase in the number of CHD as compared with younger men (P.01) (Table 3). Comet extent, % DNA in tail, and TDM were slightly increased in older men as compared with younger men, although the increase was not statistically significant. When comet assay parameters were regressed on age as a continuous measure, CHD had a statistically significant association with age (P.02); the other comet assay parameters had only small, not statistically significant, positive associations with age. For the conventional semen parameters (sperm concentration, motility, and morphology), only motility had a statistically significant inverse association with age (Table 4). TABLE 2 Distribution of comet assay parameters (n 257 patients). Minimum 5th 25th 50th 75th 95th Maximum Geometric mean Comet extent Percent DNA in tail Tail distributed moment Cells with high DNA damage Note: VisComet image analysis software was used to measure comet extent ( m), a measure of total comet length; percent DNA in tail (%), a measure of the proportion of total DNA present in the comet tail; and tail distributed moment, an integrated measure of length and intensity ( m). Cells with high DNA damage had comet tails that extended beyond the measuring boundaries in the VisComet image analysis software ( 300 m) Trisini et al. Semen and comet assay parameters Vol. 82, No. 6, December 2004

5 FIGURE 1 (A) Cell with a short comet tail. (B) Considerable heterogeneity of comet tail lengths within an individual. (C) Cell with a long comet tail. (D) Comet referred to as a cell with high DNA damage (CHD), too long to measure with the image analysis software. Scale bars, 50 m. In addition, VCL and VSL were statistically significantly lower in older men. There were no statistically significant differences in comet assay parameters for smoking status (never, current, or former smoker). Of the CASA parameters, LIN was statistically significantly lower in current smokers as compared with never smokers. However, smoking was not associated with VCL, VSL, or the conventional semen parameters. TABLE 3 Mean ( SD) of comet parameters by age and smoking status (n 257 patients). Patients (n) Comet extent ( m) % DNA in tail (%) Tail distributed moment (%) Cells with high DNA damage (number of cells) Age a (31.5) 23.4 (8.6) 58.0 (13.1) 8.0 (10.3) c (32.5) 24.4 (10.7) 60.4 (12.8) 12.6 (15.1) c Smoking b Never (30.9) 23.5 (8.6) 59.4 (12.7) 10.3 (13.0) Past (37.7) 26.8 (13.7) 58.7 (13.9) 12.9 (16.1) Current (28.8) 22.1 (8.4) 58.8 (13.4) 7.0 (6.8) a 4 patients missing age information. b 7 patients missing smoking information. c P.01. FERTILITY & STERILITY 1627

6 TABLE 4 Mean ( SD) of semen parameters by age and smoking status (n 257 patients). n Concentration ( 10 6 /ml) Motility (%) Morphology (% normal) VCL ( m/s) VSL ( m/s) LIN (%) Age a (86.2) 54.1 (23.7) c 6.42 (3.72) 79.5 (19.2) d 46.5 (10.8) c 58.4 (10.2) (103.4) 48.7 (23.1) c 7.27 (4.70) 76.8 (18.4) d 44.3 (10.8) c 57.9 (8.4) Smoking b Never (97.5) 52.3 (23.4) 6.92 (4.27) 78.1 (17.1) 45.8 (10.3) 58.9 (8.2) Past (98.8) 47.6 (24.5) 6.67 (4.19) 75.2 (23.9) 43.5 (13.3) 55.9 (13.6) Current (71.0) 52.0 (22.6) 6.77 (4.83) 84.1 (21.3) 46.3 (10.1) 56.1 (6.1) c Note: VCL curvilinear velocity; VSL straight-line velocity; LIN linearity. a 4 patients missing age information; 1 patient 35 years old missing VCL, VSL, and LIN. b 7 patients missing smoking information; 1 never-smoker patient missing VCL, VSL, and LIN. c P.05. d P.1. As expected, there were moderate to strong correlations among the comet assay parameters. Comet extent was moderately correlated with % DNA in tail and CHD (Spearman r 0.37, P.0001; r 0.47, P.0001, respectively), and strongly correlated with TDM (r 0.87, P.0001). The % DNA in tail was weakly correlated with TDM (r 0.09, P.15) and CHD (r 0.15, P.01). Finally, TDM was moderately correlated with CHD (r 0.42, P.0001). There were weak though statistically significant inverse correlations between sperm motility and comet extent (r 0.13, P.04) and % DNA in tail (r 0.14, P.03). There was also a weak correlation between sperm concentration and CE (r 0.11, P.07). The CHD had a statistically significant inverse correlation with sperm concentration (r 0.16, P.01), motility (r 0.17, P.01), VCL (r 0.14, P.02), VSL (r 0.14, P.02) and morphology (r 0.11, P.07). The TDM was not statistically significantly correlated with any of the semen parameters. In the age-adjusted regression analyses, the most consistent associations with semen parameters were found for cells with high DNA damage (Table 5). Sperm concentration, motility, morphology, VCL, VSL, and LIN were statistically significantly and negatively associated with the number of CHD (see Table 5). For an interquartile range change in CHD (IQR 10 TABLE 5 Age-adjusted regression coefficients (95% confidence intervals) for the relationship between semen parameters and comet assay parameters. Comet assay parameter Semen parameters Comet extent ( m) % DNA tail (%) Tail distributed moment ( m) Cells with high DNA damage (number of cells) Concentration ( 10 6 sperm/ml) 0.37 ( 0.73, 0.005) a 0.55 ( 1.75, 0.66) 0.53 ( 1.43, 0.37) 1.42 ( 2.30, 0.54) a Motility (% motile) 0.09 ( 0.17, 0.004) b 0.38 ( 0.67, 0.08) a 0.09 ( 0.31, 0.13) 0.43 ( 0.64, 0.21) a Morphology (% normal) 0.01 ( 0.02, 0.01) 0.05 ( 0.10, 0.01) b 0.01 ( 0.03, 0.05) 0.05 ( 0.09, 0.01) a VCL ( m/s) 0.07 ( 0.14, 0.001) b 0.19 ( 0.42, 0.05) 0.12 ( 0.30, 0.06) 0.35 ( 0.53, 0.18) a VSL ( m/s) 0.03 ( 0.07, 0.01) 0.10 ( 0.23, 0.04) 0.05 ( 0.16, 0.05) 0.19 ( 0.29, 0.09) a LIN (%) 0.03 ( 0.07, 0.004) b 0.11 ( 0.23, 0.002) b 0.07 ( 0.15, 0.02) 0.11 ( 0.19, 0.02) a Note: VCL curvilinear velocity; VSL straight-line velocity; LIN linearity. Regression coefficients: comet extent change in semen parameter/ m change in comet extent; % DNA tail change in semen parameter/% change in % DNA tail; Tail distributed moment change in semen parameter/ m change in tail distributed moment; cells with high DNA damage change in semen parameter/unit change in cells with high DNA damage. a P.05. b P Trisini et al. Semen and comet assay parameters Vol. 82, No. 6, December 2004

7 FIGURE 2 Median (25th, 75th percentiles) for comet assay parameters, plotted by semen parameters dichotomized according to WHO reference valves. TDM tail distributed moment; CHD cells with highly damaged DNA. cells), sperm concentration declined /ml, motility declined 4.3%, morphology declined 0.5%, VCL declined 3.5 m/s, VSL declined 1.9 m/s, and LIN declined 1.1%. Comet extent had a statistically significant inverse association with sperm concentration and a borderline statistically significant association with sperm motility, VCL, and LIN (see Table 5). For an interquartile range change in comet extent (IQR 42.6), sperm concentration declined /ml, sperm motility declined 3.6%, VCL declined 3.0 m/s, and LIN declined 1.3%. The % DNA tail was statistically significantly associated with sperm motility and had a borderline statistically significant association with sperm morphology and LIN. For an interquartile range change in % DNA in tail (IQR 9.1), sperm motility declined 3.4%, morphology declined 0.4%, and LIN declined 1.0%. In Figure 2 (A to D), we plotted the distribution of comet assay parameters by semen parameters categorized according to WHO reference values (15). The median comet extent, % DNA in tail, and CHD were larger for men with belowreference values for sperm concentration, motility, and morphology as compared with men above the reference values. This relationship was not as evident for TDM. In addition, the 75th percentiles for comet extent, % DNA in tail, and CHD were larger for men with semen parameters below the reference value. DISCUSSION Our study found weak though statistically significant inverse associations between some semen parameters and comet assay parameters among men visiting an andrology laboratory for evaluation of an inability to conceive with their partner. The CHD had the most consistent inverse association with semen parameters, followed by comet extent and % DNA in tail. We also found a statistically significant association between age and CHD. Interestingly, smoking status had no statistically significant association with any of the comet assay parameters. This might partially be a result of the small number of current smokers in the present study. FERTILITY & STERILITY 1629

8 The consistent associations found between CHD and age and semen parameters suggest that CHD might be a useful index for the assessment of sperm function and possibly fertility. Further exploration into the ability of CHD to predict pregnancy and pregnancy outcomes is needed before definite conclusions on its utility can be made. The agerelated increase in the number of CHD is in accordance with a previous study by Singh et al. (33), in which they observed an age-related increase in the number of DNA double-strand breaks and sperm cells with high DNA damage. Consistent with the results of our study, investigators in other human studies, using different methods to assess sperm DNA damage, also found associations between sperm DNA damage and semen parameters (7, 10, 12, 14). Benchaib et al. (7) studied 104 selected couples undergoing the assisted reproduction treatments of in vitro fertilization (IVF, n 50) and intracytoplasmic sperm injection (ICSI, n 54). They found a statistically significant inverse relationship between DNA integrity assessed with TUNEL assay and sperm concentration and motility. Using the alkaline comet assay, ISNT-condensed and ISNT-decondensed techniques, Irvine et al. (10) studied an unselected group of men (n 29) attending an infertility clinic and a small group of normozoospermic volunteer donors (n 12). They reported statistically significant inverse relations between DNA integrity and measures of semen quality, specifically sperm concentration. When comparing across the various techniques used to measure DNA integrity, they found the comet assay had the highest resolution for distinguishing between the two groups of men. Morris et al. (12) studied 60 men participating in IVF treatment, and reported a positive association between age and DNA damage measured with the alkaline comet assay. They also found a positive association between DNA damage and the percentage of sperm with abnormal morphology and a negative association between DNA damage and sperm concentration. Contrary to expectations, DNA damage was positively associated with motility, suggesting that the higher the motility the higher the DNA damage in sperm. Using TUNEL along with flow cytometry, Sun et al. (14) studied 298 men attending an infertility clinic or undergoing IVF and reported an inverse relationship between DNA damage and sperm motility, morphology, and concentration. They did not find a correlation between DNA damage and age. It is interesting that they did find evidence of a statistically significant relationship between DNA damage and current smoking. In contrast, investigators in several studies did not find consistent relationships between conventional semen parameters and measures of DNA damage (8, 9, 11, 34). These results suggested that measures of sperm DNA damage are independent of semen parameters. Chan et al. (8) studied 59 men undergoing IVF treatment, and Donnelly et al. (9) studied 17 fertile men undergoing vasectomy and 40 infertile men undergoing either IVF or ICSI. Using modified alkaline comet assay protocols, both studies reported no statistically significant correlation between sperm DNA damage and sperm morphology scored with the strict criteria. Using SCSA, Evenson et al. (11, 34) found no strong relationships between DNA damage and WHO semen parameters. Saleh et al. (13) used SCSA to assess DNA damage in 92 men seeking infertility treatment, of whom 21 had normal semen parameters and 71 had abnormal semen parameters, and in 16 fertile volunteers. The DNA damage was measured as DNA fragmentation index (% DFI), which is the percentage of cells outside the main population of t, which represents the population of cells with DNA damage. It is interesting that % DFI in sperm was statistically significantly higher in infertile men with normal semen parameters as compared with the fertile volunteers, but it was not statistically significantly different from infertile men with abnormal semen parameters. Hence, information on sperm DNA quality might provide a good explanation for idiopathic infertility in men with normal conventional semen parameters (35). Although there is overlap in mechanisms responsible for sperm DNA damage and alterations in conventional semen and CASA parameters, the weak correlations with sperm DNA damage suggest that there are differences in the sensitivity of human sperm to DNA damage and to alterations in semen and CASA parameters. In an elegant study, Aitken et al. (36) explored the relative sensitivities of sperm movement, sperm oocyte fusion, and DNA integrity to oxidative stress. At low levels of oxidative stress, DNA fragmentation, assessed with the alkaline comet assay, was statistically significantly reduced. As the level of oxidative stress increased, the sperm exhibited increased levels of DNA damage. At the highest levels of oxidative stress, sperm exhibited extremely high rates of DNA damage and loss in capacities for sperm movement and oocyte fusion. Only at the highest levels of oxidative stress did they see a decline in sperm motility. However, at lower levels of oxidative stress there were changes in sperm-movement characteristics (CASA variables), indicating that they were more sensitive to oxidative stress than percent motility. On the basis of these results, in our study we would expect to find associations between CASA parameters and comet parameters. However, contrary to expectations, CASA parameters, like the conventional semen parameters, were only statistically significantly associated with CHD. We hypothesize that cells with high DNA damage might represent cells exposed to high oxidant stress. High oxidant stress might be necessary to produce CHD, and this level of oxidant stress is also high enough to induce alterations in semen parameters, including sperm motility. The other comet assay parameters might change in response to lower levels of oxidant stress that do not alter sperm motility, and thus they were not associated with motility. We recognize 1630 Trisini et al. Semen and comet assay parameters Vol. 82, No. 6, December 2004

9 that other mechanisms apart from oxidant stress might produce CHD and alter semen parameters. Therefore, in addition to differing sensitivities to oxidative stress as a possible explanation for the weak associations found, it is also likely that there are other, as yet unknown, independent mechanisms responsible for DNA damage and altered semen parameters. Although there were statistically significant associations between conventional human semen analysis parameters and sperm DNA damage measured using the neutral comet assay, the magnitude of the associations was weak to moderate. This suggests that the assessment of sperm DNA damage provides additional independent information on sperm function and might therefore provide a complementary test to conventional semen parameters. Support for the utility of assays of DNA damage comes from studies reporting correlations between sperm DNA damage and low fertility rates for patients treated with IVF (11, 14, 37) or ICSI (11, 12, 38). In addition, Evenson et al. (39, 40) have shown that sperm DNA damage predicts fertility for men attending fertility clinics attempting to achieve pregnancy with their partners. Mechanistic studies are needed to further our understanding of the utility of measures of sperm DNA damage in the diagnostic and clinical setting. Acknowledgments: The authors thank Janna Frelich, Data Manager, and Lucille Pothier, computer programmer, at the Harvard School of Public Health, for assistance with data analysis. They also thank Linda Godfrey- Bailey, research nurse, Dr. Zuying Chen, and Nelta Mercedat Lozius, clinical laboratory assistant, at Massachusetts General Hospital, Obstetrics and Gynecology Services. References 1. Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980;210: Jurisicova A, Vermuza S, Casper RF. Programmed cell death and human embryo fragmentation. 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