Li-hong Zhang, M.Sc., a Yi Qiu, M.D., b Ke-hua Wang, M.D., Ph.D., a Qiuju Wang, M.D., c Guozhen Tao, M.D., b and Lei-guang Wang, M.D.

Transcription

1 Measurement of sperm DNA fragmentation using bright-field microscopy: comparison between sperm chromatin dispersion test and terminal uridine nickend labeling assay Li-hong Zhang, M.Sc., a Yi Qiu, M.D., b Ke-hua Wang, M.D., Ph.D., a Qiuju Wang, M.D., c Guozhen Tao, M.D., b and Lei-guang Wang, M.D. b a Department of Reproductive Medicine; b Key Laboratory for Improving Birth Outcome Technique, Shandong Research Institute for Family Planning, Jinan; and c Department of Obstetrics and Gynecology, Weifang Municipal Hospital, Weifang, People s Republic of China Objective: To compare sperm chromatin dispersion (SCD) test with terminal deoxynucleotidyle transferase-mediated terminal uridine nick-end labeling (TUNEL) assay in assessing DNA fragmentation in human sperm through bright-field microscopy. Design: Prospectively designed, side-by-side comparative study. Setting: Medical genetics laboratory in a provincial research institution. Patient(s): Sixty male patients presented for infertility evaluation and 30 fertile, volunteer sperm donors. Intervention(s): Semen analysis, SCD test, and TUNEL assay on the same semen sample and on the same spermatozoa. Main Outcome Measure(s): Sperm DNA fragmentation, determined by SCD test score or TUNEL assay score. Result(s): Sperm chromatin dispersion test and TUNEL assay identified similar proportions of sperm cells with DNA fragmentation in the same semen samples. When the SCD test and TUNEL assay were performed simultaneously on the same spermatozoa, TUNEL-negative sperm showed a large halo, whereas TUNEL-positive sperm showed no halo after Diff-Quik staining. However, in some sperm cells, DNA damage was detected by the SCD test but not by the TUNEL assay. Conclusion(s): Sperm chromatin dispersion test and TUNEL assay are both effective in detecting sperm DNA damage. Using bright-field microscopy, the SCD test appears to be more sensitive than the TUNEL assay. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Spermatozoa, DNA integrity, in situ nick-end labeling Sperm DNA fragmentation has been associated with male infertility (1 3). Poor sperm DNA integrity can negatively affect sperm fertilizing capacity in vitro (4). An accurate determination of the percentage of spermatozoa with fragmented DNA has important implications in the practice of assisted reproductive technologies (ART) because IVF using spermatozoa with damaged DNA may lead to poor embryo development or transmission of defective genetic material to the offspring (5). Sperm DNA fragmentation can be detected in a variety of ways such as the terminal uridine nick-end labeling (TUNEL) assay (6, 7), the sperm chromatin structure assay (8 10), or comet assay (11, 12). Most of these methods require complex or expensive instrumentation such as flow cytometry and fluorescence microscopy. Received February 8, 2009; revised April 10, 2009; accepted April 15, 2009; published online June 7, L.-h.Z. has nothing to disclose. Y.Q. has nothing to disclose. K.-h.W. has nothing to disclose. Q.W. has nothing to disclose. G.T. has nothing to disclose. L.-g.W. has nothing to disclose. Supported financially by the Foundation of Shandong Provincial Science and Technology Development (grant 2008GG ). Reprint requests: Li-hong Zhang, M.Sc., Department of Reproductive Medicine, Shandong Research Institute for Family Planning, 69 Yuhan Road, Jinan , People s Republic of China (FAX: ; lihong01118@126.com). The sperm chromatin dispersion (SCD) test is based on the principle that, after acid denaturing and removal of nuclear proteins, sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops, which are observed in sperm without fragmented DNA. The SCD test is a simple, less expensive method and can be performed in a short period of time. Fluorescence microscopy was originally used in the SCD test to determine sperm DNA fragmentation (13). More recently, the SCD test was modified to use bright-field microscopy (14). It was reported that the SCD test is in good concordance with the sperm chromatin structure assay. In the TUNEL assay, another test using bright-field microscopy, labeled nucleotides bind to the 3 -OH group of a broken DNA strand in the presence of terminal deoxynucleotidyle transferase. In a previous study, the SCD test using fluorescence microscopy was compared with the TUNEL assay in determining sperm DNA damage (15). Both tests yielded similar results. However, no study has been performed to compare the two using bright-field microscopy. The aims of this study are as follows: [1] to compare the SCD test with the TUNEL assay in estimating the proportion of sperm cells with DNA fragmentation in semen specimens; [2] to determine DNA fragmentation in the same spermatozoa /$36.00 Fertility and Sterility â Vol. 94, No. 3, August doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 simultaneously by the two tests; and [3] to compare the proportions of sperm with DNA fragmentation between male patients of suspected infertility and fertile men. negative control slide, and one positive control slides. The fourth slide was prepared for performing SCD and TUNEL assay simultaneously. MATERIALS AND METHODS Patients and Semen Analysis Semen samples were obtained from male partners of infertile couples presenting to the Fertility Center of Shandong Research Institute for Family Planning for evaluation (n ¼ 60) and from fertile, volunteer sperm donors (n ¼ 30). Infertile couples were those who failed to conceive after unprotected intercourse for at least 1 year. All of the volunteer donors had previously fathered at least one child. Samples were produced by masturbation after 2 3 days of sexual abstinence and were allowed to liquefy at room temperature. After liquefaction, standard semen analysis was performed on the semen samples, according to the World Health Organization (WHO) guidelines (16), to record volume, sperm concentration, sperm motility, and sperm morphology. Sperm morphology was assessed using Tygerberg s strict criteria after Diff-Quik staining (17). Information on the semen source (patients or fertile volunteers) was withheld from technologists who performed semen analyses and DNA fragmentation testing. None of the semen samples had significant leukocytospermia as per WHO guidelines (<1 million round cells per milliliter). Identification information for all subjects in this study was held confidential and protected from the public. This study was approved by the internal review board of Human Ethics at Shandong Research Institute for Family Planning, China. Semen Processing for SCD Test and TUNEL Assay Semen sample was mixed with 5 6 ml of modified human tubal fluid (mhtf) medium (Irvine Scientific, Santa Ana, CA) in a centrifuge tube to achieve a ratio of at least 2 parts medium to 1 part semen. After gently inverting the centrifuge tube three to five times, the sample was then centrifuged for 10 minutes at 300 g. After the second wash, the supernatant was removed, and the sperm pellet was resuspended in mhtf medium at a concentration of spermatozoa per milliliter and aliquoted for DNA damage testing by SCD or TUNEL assay. All samples were processed within 1.5 hours after they were produced. An aliquot of the washed sperm sample was mixed with 1% (wt/vol) low melting point agarose (to obtain a 0.7% final agarose concentration) at 37 C. Aliquots of 50 ml of the mixture were pipetted onto the glass microscopic slides precoated with 0.65% agarose, covered with a cover slip (24 60 mm), and left to solidify at 4 C for 5 minutes. Cover slips were carefully removed from slides. Among these slides, three slides, including one TUNEL slide, one negative control slide, and one positive control slide, were fixed in 4% paraformaldehyde at room temperature, and stored at 4 C for up to 4 weeks for TUNEL assay. Four slides were prepared for SCD test, including one SCD test slide, one SCD Test The SCD test was performed following the procedure of Fernandez et al. (13), with minor modifications. After removing cover slips from solidified samples, the slides were immediately immersed horizontally in a tray with freshly prepared acid denaturing solution (0.08 N HCl) for 7 minutes at 22 C in the dark to generate restricted single-stranded DNA (ssdna) motifs from DNA breaks. The denaturing was then stopped and proteins were removed by transferring the slides to a tray with neutralizing and lysis solution (0.4 M Tris, 0.4 M 1,4-dithiothreitol [DTT], 1% Triton X-100, and 50 mm ethylenediaminetetraacetic acid [EDTA], ph 7.5) for 15 minutes 22 C, which was followed by washing in Tris-borate-EDTA buffer (0.09 M Tris borate and M EDTA, ph 7.5) for 2 minutes; dehydrated in sequential 70%, 90%, and 100% ethanol baths (2 minutes each); and air dried. Slides may be stored at room temperature for several months in a tightly closed box in the dark. Then the slides can be stained with the Diff-Quik reagent for bright-field microscopy, or incubated with terminal deoxynucleotidyle transferase enzyme for TUNEL assay. A total of 500 sperms were evaluated manually on each slide for halo size and dispersion pattern as described by Fernandez et al. (14): [1] nuclei with large DNA dispersion halos, [2] nuclei with medium-sized halos, [3] nuclei with small-sized halos, and [4] nuclei with no halo (Fig. 1). The nuclei with large-to-medium size halo were considered sperm with nonfragmented DNA, whereas nuclei with small size halo or without halo or without a halo and degraded were considered sperm with fragmented DNA. TUNEL Assay The TUNEL assay was performed using the In-Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany). The fixed slides were rinsed in phosphate-buffered solution (PBS), ph 7.4, and then permeated with 2% Triton X-100. The terminal deoxynucleotidyle transferase-labeled nucleotide mixture was added to each slide and incubated in a humidified chamber at 37 C for 60 minutes in the dark. Later, slides were rinsed three times in PBS for 5 minutes each. Converter-peroxidase solution (POD) was added on sample. The slides were incubated in a humidified chamber for 30 minutes at 37 C, and rinsed three times in PBS, and incubated in the presence of diaminobenzidine substrate for 10 minutes at C. The slides were further rinsed three times with PBS and counterstained with 2% methyl green. A total of 500 sperm per individual were examined by the same examiner using bright-field microscopy. The total number of sperm per field, stained with methyl green, was 1028 Zhang et al. Sperm DNA fragmentation Vol. 94, No. 3, August 2010

3 FIGURE 1 The sperm chromatin dispersion test: sperm with different size halos. Sperms (a) show the nuclei with large size halo and (b) show the nuclei with medium size halo that were considered with nonfragmented DNA, whereas (c) show the nuclei with small size halo, (d) show without halo, (e) show without a halo and degraded, and (f) show without halo and pinhead, which were considered sperm with fragmented DNA. 1 U/mL for 20 minutes at room temperature) controls were performed in each experiment. The final percentage of sperm with fragmented DNA was referred to as percentage of TUNEL-positive sperm. Simultaneous SCD Test and TUNEL Assay on the Same Spermatozoa Immediately after SCD, and on the same slide, TUNEL assay was performed to detect DNA fragmentation. Sperm DNA fragmentation was assessed in several randomly selected fields under bright-field microscopy using a 1,000 oil immersion objective. A total of 200 cells were evaluated, in two aliquots per patient. During this examination, every time a TUNELpositive sperm was found, an image of the field was captured with a digital camera. Immediately, Diff-Quik reagents were added and the image of the same field with stained spermatozoa was captured again. The halo of TUNEL-positive sperm and the halo of TUNEL-negative sperm were then examined by comparing the images captured before and after staining. Statistical Analysis All results were expressed as mean SD. Data were analyzed with Student s t-test, analysis of variance (ANOVA), or linear regression, using SPSS 11.0 software (SPSS, Chicago, IL). All hypothesis testing was two-sided with the P value of less than.05 considered statistically significant. counted first. The number of brown cells (TUNEL positive) was then counted and expressed as a percentage of the total sperm cells. Negative (omitting the enzyme terminal transferase) and positive (incubation with deoxyribonuclease I, RESULTS The means and SD for the basic seminal parameters of male patients and fertile volunteers are summarized in Table 1. The sperm concentration, forward motility, and normal morphology are lower in patients than in fertile volunteers. The number of sperm with DNA fragmentation is significantly TABLE 1 Seminal characteristics of infertile patients and fertile men. Fertile men (n [ 30) Infertile patients (n [ 60) Total (n [ 90) Basic seminal parameters Age (y) Sperm concentration (10 6 /ml) d Forward motility (%) d Normal morphology (%) d Sperm DNA fragmentation (%) SCD test a d,b c TUNEL assay d Note: Values are mean SD. a P¼.155, compared with TUNEL assay. b P¼.001, compared with TUNEL assay. c P¼.000, compared with TUNEL assay. d P<.001, Compared with fertile group. Fertility and Sterility â 1029

4 FIGURE 2 The sperm chromatin dispersion test and the terminal uridine nick-end labeling (TUNEL) assay on the same slide (before and after Diff-Quik staining). (A) Before Diff-Quik staining, TUNEL-positive sperms were showed stained brown. (B) After Diff-Quik staining, TUNEL-positive sperms showed no halo and TUNEL-negative sperms showed a large halo. No halo pinhead sperm (arrow) could not be found by TUNEL assay before Diff-Quik staining. higher in male patients than in fertile volunteers as assessed by both SCD test and TUNEL assay. The percentage of sperm with fragmented DNA in male patients and in fertile volunteers were 25.2% 10.2% and 13.6% 3.4%, respectively, as assessed by SCD test (P<.001), and 22.8% 6.9% and 12.9% 3.0%, respectively, by TUNEL assay (P<.001). Sperm chromatin dispersion test detected a significantly higher proportion of sperm with fragmented DNA than the TUNEL assay did (21.4% 10.1% vs. 19.5% 7.5%; P<.001). When infertility patients and fertile volunteers are considered separately, the difference in the results from the two tests was statistically significant only for the male patients (25.2% 10.2% from SCD vs. 22.8% 6.9%; P<.001), but not for fertile men (13.6% 3.4% vs. 12.9% 3.0%; P>.1). When the SCD test and TUNEL assay were performed simultaneously on the same sample slide, TUNEL-negative sperm show a large halo and TUNEL-positive sperm (brown cell) show no halo after Diff-Quik staining. Some type of sperm, such as pinhead sperm, could not be shown before Diff-Quik staining. After Diff-Quik staining, pinhead sperm and some degrade sperm could be seen clearly (Fig. 2). The results of the SCD test and TUNEL assay exhibited a statistically significant correlation for sperm DNA fragmentation within infertile patients and within fertile men (r ¼ 0.876, P<.001; r ¼ 0.627, P<.001). The linear relationships were weak between sperm concentration and sperm DNA fragmentation (r ¼ in SCD test, r ¼ in TUNEL assay), and normal morphology and sperm DNA fragmentation (r ¼ in SCD test, r ¼ in TUNEL assay). However, the linear relationship between forward motility and sperm DNA fragmentation was moderate (r ¼ in SCD test, r ¼ in TUNEL assay). These three semen analysis parameters were all negatively associated with sperm DNA 1030 Zhang et al. Sperm DNA fragmentation Vol. 94, No. 3, August 2010

5 TABLE 2 Univariate linear regression and multiple linear regression analysis for factors of sperm DNA fragmentation. Seminal factors Sperm DNA fragmentation SCD test TUNEL assay Univariate linear regression Sperm concentration a a Forward motility a a Normal morphology a a Multiple linear regression Sperm concentration Forward motility a a Normal morphology Note: The number in the cells of univariate regression model are correlation coefficient (r values); the number in the cells of multiple regression model are standardized coefficients (b values). a P<.001. fragmentation (P<.001). However, in multiple regression analysis, after controlling for the effect of the other two factors, forward motility still maintains the negative association with sperm DNA fragmentation. When forward motility increases one unit, the sperm DNA fragmentation will decrease 0.41 units. The association between sperm concentration and sperm DNA fragmentation and the association between normal morphology and sperm DNA fragmentation disappeared, after controlling for the effects of sperm concentration and normal morphology on sperm DNA fragmentation (Table 2). DISCUSSION This study demonstrated for the first time that, when SCD test and TUNEL assay were performed on the same slide through bright-field microscopy, TUNEL-negative spermatozoa displayed a large halo, indicative of intact DNA. No halo was observed in TUNEL-positive sperm after Diff-Quik staining. Spermatozoa identified by SCD test as containing fragmented DNA were also positively stained with the TUNEL assay. Among the reported studies on biochemical markers of male infertility (18 20), much attention has been given to sperm DNA fragmentation. DNA damage may be caused by defective chromatin packaging during spermiogenesis, apoptosis during either spermatogenesis or sperm transport through the male genital tract (21), or as a consequence of oxidative stress (22). Fernandez et al. (13) reported the use of breakage detection fluorescence in situ hybridization in the SCD test for sperm DNA fragmentation. Spermatozoa with a very small or absent halo showed extensive DNA fragmentation by breakage detection fluorescence in situ hybridization. The sperm chromatin dispersion test is a relatively new technique to assess sperm DNA fragmentation. The previous study (14) demonstrated that the halo size, which may represent the degree of nuclear DNA damage, could be estimated with a conventional bright-field microscope using the Wright stain or the Diff-Quik stain. Results obtained from the SCD test correlate well with those from other DNA integrity tests, such as sperm chromatin structure assay and TUNEL (14, 15). Unlike the sperm chromatin structure assay and TUNEL, SCD can be performed without complex or expensive instrumentation such as flow cytometry or fluorescence microscopy. The TUNEL assay has been widely used to identify apoptotic cells in tissue sections. Studies confirmed that the TUNEL assay was an accurate method for quantifying apoptosis in germ cells (6, 23, 24). The use of biotinylated nucleotides in the TUNEL assay, coupled with signal amplification by the avidin-peroxidase reaction, allows signal detection through bright-field microscopy, as demonstrated in this study. This study confirmed the findings of Chohan et al. (15) that a strong relationship exits between the SCD test and TUNEL assay (Table 1). The present study also demonstrated that the SCD test identified more spermatozoa with DNA damage than the TUNEL assay could for the male partners of infertile couples, suggesting that the SCD test may be more sensitive. This result was inconsistent with the results from a previous study (15), which found that the SCD test and TUNEL assay have similar sensitivity. Patient selection criteria may be one of the reasons to explain these contradictory conclusions. Another possible reason is that the SCD test is more accurate using microscopy than using fluorescence microscopy, which could not distinguish spermatozoa from other cell types due to its inability to visualize sperm tails (14). By simultaneously performing the SCD test and TUNEL assay, we observed that some sperm, such as pinhead sperm, could not be visualized before Diff-Quik staining. After Diff- Quik staining, pinhead sperm and some degraded sperm could be observed clearly. In addition, the sperm tails remain intact with the SCD test, allowing easy determination of sperm cells from other cell types present in the sample. We found that male partners of infertile couples have a significantly higher percentage of sperm with fragmented DNA compared with fertile men. These results are in agreement with previous findings (25). Earlier studies also found that infertile men have more spermatozoa with abnormal morphology than fertile men (26, 27). The univariate regression analysis in the present study is also in agreement with other studies in which sperm DNA fragmentation was negatively correlated with conventional semen analysis parameters such as sperm concentration, progressive motility, and morphology (5, 28, 29). However, using multiple linear regression analysis, we found that only sperm motility was a statistically significant predictor of the proportion of the sperm with DNA fragmentation as assessed by both the SCD test and TUNEL assay (Table 2). This Fertility and Sterility â 1031

6 was inconsistence with the results from a previous study showing that neither sperm concentration, motility, nor morphology were statistically significant predictors of the proportions of sperm cells with DNA strand breaks as assessed by TUNEL analysis (28). Whether or not the sperm DNA integrity is an independent parameter for sperm DNA integrity should be confirmed in additional studies. In conclusion, both the SCD test and TUNEL assay are effective and simple methods for detecting sperm DNA fragmentation, but the SCD test appears to be more sensitive. Acknowledgments: The authors thank Qi Zhang, Juan Li, Xinying Li, and Jianchun Yu for their help on the study. Thanks also go to Dr. Fabin Han for assistance with manuscript editing and Dr. John Zhang (IVF Laboratory, Department of Obstetrics & Gynecology, Feinberg School of Medicine, Northwestern University) for the linguistic revision of the final version of the manuscript. REFERENCES 1. Erenpreiss J, Elzanaty S, Giwercman A. Sperm DNA damage in men from infertile couples. Asian J Androl 2008;10: Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999;14: Zini A, Kamal K, Phang D, Willis J, Jarvi K. Biologic variability of sperm DNA denaturation in infertile men. Urology 2001;58: Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, et al. Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril 2004;81: Benchaib M, Lornage J, Mazoyer C, Lejeune H, Salle B, Francxois Guerin J. Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome. Fertil Steril 2007;87: Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells. Exp Cell Res 1993;207: Piasecka M, Gaczarzewicz D, Laszczynska M, Starczewski A, Brodowska A. Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia. Folia Histochem Cytobiol 2007;45(Suppl 1):S Evenson D, Jost L. Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci 2000;22: Bungum M, Spano M, Humaidan P, Eleuteri P, Rescia M, Giwercman A. Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART. Hum Reprod 2008;23: Alvarez JG, Lewis S. Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive of the outcome of ART. Hum Reprod 2008;23: Migliore L, Naccarati A, Zanello A, Scarpato R, Bramanti L, Mariani M. Assessment of sperm DNA integrity in workers exposed to styrene. Hum Reprod 2002;17: Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002;17: Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez JG. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J Androl 2003;24: Fernandez JL, Muriel L, Goyanes V, Segrelles E, Gosalvez J, Enciso M, et al. Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test. Fertil Steril 2005;84: Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl 2006;27: World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge, UK: Cambridge University Press, Van Waart J, Kruger TF, Lombard CJ, Ombelet W. Predictive value of normal sperm morphology in intrauterine insemination (IUI): a structured literature review. Hum Reprod Update 2001;7: Agarwal A, Sharma RK, Nallella KP, Thomas AJ Jr, Alvarez JG, Sikka SC. Reactive oxygen species as an independent marker of male factor infertility. Fertil Steril 2006;86: Khalili MA, Aghaie-Maybodi F, Anvari M, Talebi AR. Sperm nuclear DNA in ejaculates of fertile and infertile men: correlation with semen parameters. Urol J 2006;3: Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity tests predict pregnancy with in vitro fertilization? Fertil Steril 2008;89: Duran EH, Gurgan T, Gunalp S, Enginsu ME, Yarali H, Ayhan A. A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro. Hum Reprod 1998;13: Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998;59: Baccetti B, Collodel G, Piomboni P. Apoptosis in human ejaculated sperm cells (notulae seminologicae 9). J Submicrosc Cytol Pathol 1996;28: Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000;21: Zini A, Fischer MA, Sharir S, Shayegan B, Phang D, Jarvi K. Prevalence of abnormal sperm DNA denaturation in fertile and infertile men. Urology 2002;60: Gunalp S, Onculoglu C, Gurgan T, Kruger TF, Lombard CJ. A study of semen parameters with emphasis on sperm morphology in a fertile population: an attempt to develop clinical thresholds. Hum Reprod 2001;16: Guzick DS, Overstreet JW, Factor-Litvak P, Brazil CK, Nakajima ST, Coutifaris C, et al. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med 2001;345: Erenpreiss J, Jepson K, Giwercman A, Tsarev I, Erenpreisa J, Spano M. Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod 2004;19: Muratori M, Marchiani S, Tamburrino L, Tocci V, Failli P, Forti G, et al. Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters. Hum Reprod 2008;23: Zhang et al. Sperm DNA fragmentation Vol. 94, No. 3, August 2010