Clinical value of sperm DNA damage should be assessed in motile sperm fraction rather than whole ejaculated sperm

Clinical value of sperm DNA damage should be assessed in motile sperm fraction rather than whole ejaculated sperm De-Yi Liu, Ph.D., a,b and Ming-Li Liu, B.Sc. a a Melbourne IVF and b Department of Obstetrics and Gynaecology (Royal Women's Hospital), University of Melbourne, Melbourne, Victoria, Australia Objective: To determine differences and frequency of excessive (R50%) sperm DNA damage between ejaculated and motile sperm. Design: Sperm DNA damage was assessed by acridine orange fluorescence and the results of ejaculated and motile sperm were compared. Setting: Public and private clinical assisted reproduction centers. Patient(s): A total of 272 subfertile men were studied. Intervention(s): None. Main Outcome Measure(s): Semen analysis and sperm DNA damage. Result(s): Sperm DNA damage was negatively correlated with sperm motility and normal morphology. Overall, 39.7% (108 of 272) of semen samples had excessive sperm DNA damage. In contrast, only 15% (41 of 272) of motile sperm fractions had excessive DNA damage. Based on DNA results of motile sperm and semen characteristics, the proportion of men with excessive sperm DNA damage was 26% in severe teratozoospermia, 17.5% in oligozoospermia, 12.5% in moderate teratozoospermia, and 4.6% in normozoospermia. Severe teratozoospermia had five times more frequent excessive DNA damage than normozoospermia. Conclusion(s): Abnormal sperm morphology is highly associated with sperm DNA damage. Results of DNA damage of ejaculated sperm do not accurately reflect DNA status of motile sperm. Therefore, sperm DNA damage should be assessed in motile sperm fraction rather than whole ejaculated sperm. (Fertil Steril Ò 2013;99:367 71. Ó2013 by American Society for Reproductive Medicine.) Key Words: Male infertility, semen quality, DNA damage of motile sperm Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/liudy-sperm-dna-damage-male-infertility/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. In the literature, there are many reports on the relationship between fertilization and pregnancy rates in clinical assisted reproductive technologies (ART) and sperm DNA damage detected with the use of various methods, including acridine orange (AO) fluorescence stain assessed either by fluorescent microscope or flow cytometry (sperm chromatin structure assay), sperm chromatin dispersion, terminal deoxynucleotidyl transferase mediated dudp nick-end labeling or electrophoresis (Comet assays). Results of sperm DNA damage obtained from these different methods are highly correlated with each other. The AO fluorescence stain distinguishes sperm with either double- (green fluorescence) or single-stranded (or denatured, red fluorescence) DNA. The proportion of ejaculated sperm with abnormal DNA Received June 28, 2012; revised August 26, 2012; accepted October 2, 2012; published online October 22, 2012. D.-Y.L. has nothing to disclose. M.-L.L. has nothing to disclose. Reprint requests: De-Yi Liu, Ph.D., Melbourne IVF, 344 Victoria Parade, East Melbourne 3002, Victoria, Australia (E-mail: deyi.liu@mivf.com.au). Fertility and Sterility Vol. 99, No. 2, February 2013 0015-0282/$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.10.005 detected by AO is negatively correlated with fertilization rates in in vitro fertilization (IVF) or pregnancy rates in IVF and intracytoplasmic sperm injection (ICSI) (1 14). However, many studies have found no or weak correlations between sperm DNA damage and the outcomes of IVF and ICSI (15 19). Therefore, clinical value of tests for sperm DNA damage is still not conclusive. Currently, one of the major problems is that assessment of sperm DNA damage is mostly performed in raw semen and not in motile sperm fraction. It is known that semen contains a mixture of motile, immotile, nonviable, and degenerated sperm and results of sperm DNA damage in semen detected mainly VOL. 99 NO. 2 / FEBRUARY 2013 367

ORIGINAL ARTICLE: ANDROLOGY reflect the nonviable sperm and not accurately the motile sperm fraction. In human fertilization either in vivo or in vitro, only motile sperm have a chance to fertilize an oocyte. Therefore it is physiologically logical to screen DNA damage in motile sperm fraction and not in raw semen. Although many studies showed clearly that motile sperm selected by either swim-up or colloidal silica gradient centrifugation had significantly lower proportion of DNA damage than sperm in raw semen (2, 15, 20, 21), the majority of clinical data on the relationship between sperm DNA damage and the outcome of IVF/ICSI are based only on the results of ejaculated sperm and not motile sperm fraction. Thus, there is a lack of clinical information on the difference between sperm DNA results obtained from semen and motile sperm fraction. In the present study, we report the frequency of excessive sperm DNA damage in both ejaculated sperm and selected motile fraction in 272 subfertile men. MATERIALS AND METHODS Subjects and Semen Analysis Semen samples were obtained by masturbation after an average of 3 days' abstinence from 272 subfertile men (no natural conception after the couples had tried R12 months) who attended our infertility clinics in both the Royal Women's Hospital and Melbourne IVF. Men with severe oligozoospermia or asthnozoospermia with <0.2 million motile sperm recovered after sperm preparation were excluded from the study. Routine semen analysis was performed after liquefaction of the semen within 2 hours according to the fourth-edition World Health Organization manual (22). The percentage of sperm motility and progressive motility were assessed manually by counting 400 sperm. Sperm morphology was assessed on smears prepared by washing of sperm in 10 ml 0.9% sodium chloride. Washing sperm to remove seminal plasma or protein in medium decreases background of staining and produces clearer images of sperm. The percentage of normal sperm morphology was assessed according to strict criteria (22). For each sperm sample, 200 spermatozoa were scored from at least ten individual fields with the use of oil immersion and magnification of 1,000 under bright-field illumination. All morphology slides were assessed by two scientists independently, and the mean of the results was used. The Royal Women Hospital Research and Ethics Committees approved the project. Sperm Preparation The same ejaculate was split into two parts: One part was directly used to prepare sperm smear for DNA test, and the other part was used to harvest motile sperm fraction by colloidal silica gradient centrifugation (Puresperm; Nidacon International) with the use of two layers of 1 ml 40% and 1 ml 80% Puresperm. The pellet of motile sperm obtained from Puresperm preparation was washed once with 1 ml human tubal fluid (HTF; Irvine Scientific) supplemented with 2% human albumin (Irvine Scientific). The washed sperm pellet was resuspended with the same HTF medium to a sperm concentration of 2 10 6 /ml and then left it in the 5% CO 2 incubator for 2 hours to allow sperm equilibration from sperm preparation procedures. Acridine Orange Fluorescence for Sperm Chromatin DNA The sperm chromatin DNA damage was assessed by the AO (Sigma Chemical Co.) fluorescence method described by Tejada et al (23). Briefly, after air-drying the sperm smears, the smears were fixed in Carnoy solution (3 parts methanol and 1 part glacial acetic acid) for 3 hours or overnight at 4 C. After fixation, the slides were allowed to dry in air for a few minutes before staining with AO solution. The AO staining solution was prepared daily as follows: 2.5 ml 1% AO stock solution in distilled water was added to a mixture of 10 ml 0.1 mol/l citric acid and 0.63 ml 0.3 mol/l Na 2 H- PO 4 7H 2 O, ph 2.5. The 1% AO stock solution was stored in the dark at 4 C for 2 weeks. The fixed and air dried sperm smear was stained in the AO work solution for 5 minutes and then the slide was gently rinsed and mounted with distilled water. The percentage of sperm with damaged DNA was determined by randomly scoring 200 sperm under a fluorescence microscope (Dialux 20; Leitz) with 400 magnification and excitation of 450 490 nm. Sperm with normal (double-stranded) DNA were fluorescence green, and those with damaged (denatured or single stranded DNA) were fluorescence red or yellow. To obtain consistent results from this procedure, duplicate slides were made for each sperm sample and assessed by two experienced observers independently, and the mean of the two results was used. Statistical Analysis The significance of correlations between semen analysis results and sperm DNA damage in raw semen and motile sperm fraction were examined by Spearman tests. Differences of sperm DNA results between ejaculated sperm and motile population were examined by nonparametric Wilcoxon test. The proportion of men with excessive (R50%) sperm DNA damage between ejaculated and motile sperm were compared by chi-square test. RESULTS There was a wide range for all of the sperm test results, including sperm DNA damage, and the results of DNA damage of ejaculated sperm was significantly higher than those of motile sperm fraction (Table 1). All semen analysis results were significantly correlated with sperm DNA damage (Table 2). Overall sperm morphology was the most significantly correlated with sperm DNA damage (Fig. 1). Based on DNA results of ejaculated sperm, 39.7% (108 of 272) of semen samples had excessive (R50%) DNA damage detected. In contrast, only 15% (41 of 272) of motile sperm fractions had excessive sperm DNA damage detected. When the same data were analyzed according to seminal characteristics as oligozoospermia (<20 10 6 /ml; n ¼ 40) and men with sperm concentration >20 million/ml but normal sperm morphology %5% (severe teratozoospermia; n ¼ 88), 6% 9% (moderate teratozoospermia; n ¼ 56) and R10% (normozoospermia; n ¼ 88), the proportion of men with excessive (R50%) sperm DNA damage ranged from 9.8% to 62.5% according to the results of ejaculated sperm but only 4.6% to 26% according to the results of motile sperm fraction 368 VOL. 99 NO. 2 / FEBRUARY 2013

Fertility and Sterility TABLE 1 FIGURE 1 Age of men and all sperm tests results in 272 subfertile men. Variable Mean ± SD Range Age of men (y) 37.1 5.6 25 50 Days of abstinence 3.7 2.0 1 20 Semen volume (ml) 3.7 2.0 0.6 9.2 Sperm concentration (10 6 /ml) 81.3 76.9 2 333 Total motility (%) 52.7 12.4 19 83 Progressive motility (%) 43.6 13.7 7 76 Normal sperm morphology (%, semen) 9.2 6.9 0 39 DNA-damaged sperm (%, semen)* 44.9 18.7 11 88 DNA-damaged sperm (%, motile)* 31.9 17.1 4 81 * Comparison between semen and motile sperm: P<.001. (Fig. 2). The proportion of men with excessive (R50%) DNA damage of motile sperm was the highest in severe teratozoospermia (26%), then oligozoospermia (17.5%), moderate teratozoospermia (12.5%), and the lowest in normozoospermia (4.6%; Fig. 2). Thus excessive (R50%) DNA damage of motile sperm in normozoospermic subfertile men was only 4.6% which was about five times less frequent than severe teratozoospermic men (26%). Overall, there was significant reduction (two to three times) in the proportion of men with excessive DNA damage when sperm DNA was assessed in motile sperm fraction compared with the results of whole ejaculated sperm. DISCUSSION The present study shows clearly that results of DNA damage of ejaculated sperm do not reflect actual DNA status of motile sperm fraction. Based on the results of ejaculated sperm, frequency of excessive (R50%) sperm DNA damage was about three times higher than the results of motile sperm fraction on the same samples from 272 subfertile men. Clearly, raw semen contains a large number of nonviable or degenerated sperm which directly contributes to high DNA damage results. Because only motile or live (for ICSI only) sperm can fertilize an oocyte in vivo or vitro including IVF and ICSI, it is logical (A) Correlation between sperm morphology in semen and sperm DNA damage in semen (Spearman r ¼ 0.401; n ¼ 272; P<.001); (B) correlation between sperm morphology in semen and sperm DNA damage in motile fraction (Spearman r ¼ 0.309; P<.001). TABLE 2 Correlations (Spearman test) between sperm DNA damage in semen or motile population and age of men and semen analysis results. Test DNA damage (semen) DNA damage (motile) Coefficient P value Coefficient P value Age of male (y) 0.159 <.01 0.051.398 Days of abstinence 0.046 >.05 0.039 >.05 Semen volume (ml) 0.135 <.05 0.151 <.01 Sperm concentration 0.192 <.005 0.137 <.005 (10 6 /ml) Total motility (%) 0.276 <.001 0.142 <.001 Progressive motility (%) 0.341 <.0001 0.170 <.001 Normal sperm 0.400 <.0001 0.309 <.0001 morphology (%) DNA damage (semen, %) 0.774 <.0001 that nonviable sperm should be excluded from assessment of sperm DNA damage. This may explain why some earlier studies found that results of DNA damage assessed in raw semen was not highly correlated with IVF fertilization rates or IVF/ ICSI pregnancy rates (13, 16 19). In the present study, the proportion of men with excessive (R50%) sperm DNA damage in motile sperm fraction was 26%, 18%, 12%, and 4.6% for severe teratozoospermia (normal morphology %5%), oligozoospermia, moderate teratozoospermia (normal morphology 6% 9%), and normozoospermia (normal morphology R10%), respectively. Thus both severe teratozoospermic and oligozoospermic subfertile men had a very high incidence of excessive DNA damage of motile sperm. In contrast, only <5% of normozoospermic subfertile men had excessive DNA damage of motile sperm. Therefore, it is clinically more critical to screen DNA damage VOL. 99 NO. 2 / FEBRUARY 2013 369

ORIGINAL ARTICLE: ANDROLOGY FIGURE 2 Comparison of the proportion of men with excessive (>50%) DNA damage obtained between raw semen and motile sperm fraction in subfertile men with oligozoospemia (P<.01) or normozoospermia with various normal sperm morphology (P<.001.05). of motile sperm in both oligozoospermic and severe teratozoospermic men. It is very important to diagnose those patients with excessive (R50%) DNA damage of motile sperm before they commence ICSI treatment, so an alternative sperm selection procedure can be applied to enhance the chance of selecting a normal DNA sperm for ICSI to achieve better outcome. In the literature, several alternative sperm selection procedures have been reported. Using high-magnification microscopy combined with computer-enlarged image for selection of morphologically normal sperm with less DNA damage may improve ICSI outcomes (24, 25). It is also reported that sperm bound to the solid phase of hyaluronic acid (HA) have better morphology and less DNA damage, which may improve sperm selection for ICSI, but there are insufficient data to confirm if using HA-bound sperm for ICSI can improve implantation and pregnancy (26, 27). Physiologically, the human zona pellucida (ZP) has the capacity to bind selectively to sperm with normal morphology and normal DNA, and only an average of 14% of motile sperm from ejaculate of fertile men is capable of binding to the ZP in vitro (28, 29). Therefore, it is possible to use the ZP-binding process to select normal DNA sperm for ICSI. There were three independent studies showing that using patients' sibling immature oocyte to prepare ZPbound sperm for ICSI significantly enhanced embryo quality, implantation, and pregnancy rates (30 33). However, more clinical studies with a larger number of subjects are needed to confirm if any of the above alternative sperm selection methods are clinically useful to select a normal DNA sperm for ICSI in patients diagnosed with excessive sperm DNA damage. In the present study we used strict morphology assessment described in fourth edition of the World Health Organization manual (22). We confirmed that there is a high correlation between normal sperm morphology and normal chromatin DNA in both semen and motile sperm (1, 4, 6, 15, 20, 32). This indicates that sperm morphologic defects are highly associated with sperm DNA damage, which may reflect sperm immaturity or abnormal spermatogenesis. Despite the high association between abnormal sperm morphology and DNA damage, in some infertile men a high proportion of morphologically normal sperm had DNA damage detected (34, 35). However, results of the present study showed that only <5% of normozoospermic (normal sperm morphology R10%) subfertile men had excessive DNA damage of motile sperm. In this study, we did not have clinical data on the ART outcome for all of the men. Therefore, further study is needed to determine if sperm DNA damage assessed in motile sperm fraction can provide better predictive value for the ART outcomes than the DNA results of ejaculated sperm. In conclusion, raw semen contains a large number of nonviable and degenerated sperm with damaged DNA, so the results of DNA damage of ejaculated sperm do not accurately reflect DNA status of motile sperm fraction. Therefore, clinical assessment of sperm DNA damage should be performed in motile sperm fraction rather than whole ejaculated sperm. However, further study is required to determine if DNA damage results of motile sperm fraction will provide a better predictive value for male fertility or IVF/ICSI outcomes. 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