- Email: [email protected]
Flow cytometric measurement of sperm nuclear DNA fragmentation in infertile men with normal standard sperm parameters
Original article
Keywords Sperm DNA fragmentation Flow cytometry Male infertility Sperm
Tzvetan H. Lukanov, MD, PhD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, and Center for Reproductive Health, Medical University of Pleven 1, Pleven, Bulgaria Danail I. Lichev, MD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, and Center for Reproductive Health, Medical University of Pleven, Pleven, Bulgaria Emiliana I. Konova, MD, PhD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, and Center for Reproductive Health, Medical University of Pleven, Pleven, Bulgaria Alkan I. Emin, MD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, and Center for Reproductive Health, Medical University of Pleven, Pleven, Bulgaria Nina P. Ayvazova Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, Bulgaria Anjelika V. Velkova, MD, PhD Department of Social Medicine, Medical University of Pleven, Pleven, Bulgaria Roumen G. Roussev, MD, PhD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, Bulgaria and Millenova Immunology Laboratories, Chicago, Illinois, USA
Flow cytometric measurement of sperm nuclear DNA fragmentation in infertile men with normal standard sperm parameters Tzvetan H. Lukanov, Danail I. Lichev, Emiliana I. Konova, Alkan I. Emin, Nina P. Ayvazova, Anjelika V. Velkova and Roumen G. Roussev Abstract Background: Male-factor infertility plays a role in approximately 50% of infertile couples. In at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results. When diagnostic work-up of the female partner is also normal, they are classified as idiopathic. The objective of this study was to evaluate the levels of sperm nuclear DNA fragmentation in a population of infertile men with normal standard semen parameters and to compare their results with those from men who had abnormal semen parameters, as well as with a control group of fertile men. Methods: Semen samples were obtained from 202 infertile men and 30 fertile donors. Standard semen analysis was performed according to the World Health Organization guidelines. Flow cytometry has been extensively used to study sperm DNA fragmentation and the results are expressed as the percentage of sperm DNA fragmentation index (DFI). Results: Of the 202 patients, 48 (23.8%) had normal standard sperm parameters, while 154 (76.2%) had an abnormality in one or more of these parameters. DFI in infertile men with normal sperm parameters was significantly higher than in fertile donors (p = 0.03), but not significantly different from infertile men with abnormal sperm parameters (p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperm from infertile men with abnormal and normal semen parameters, but not in fertile donors (r = 0.26, p = 0.001 and r = 0.48, p = 0.0001, respectively). Conclusion: Sperm from infertile men with normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to levels in infertile men with abnormal sperm parameters. Sperm DNA fragmentation analysis is an independent test of sperm quality and has an important diagnostic value in the evaluation of male infertility. ß 2008 WPMH GmbH. Published by Elsevier Ireland Ltd.
Introduction
E-mail: [email protected]
Online 20 November 2008
50
Male-factor infertility plays a role in approximately 50% of infertile couples [1]. Standard
Vol. 6, No. 1, pp. 50–55, March 2009
semen analysis consists of measuring a variety of semen parameters, including volume, pH, sperm concentration, vitality, and morphology, the presence of leukocytes and immature
ß 2008 WPMH GmbH. Published by Elsevier Ireland Ltd.
Original article germ cells, and sperm motility [2]. 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, diagnosing defective sperm function by conventional semen analysis is difficult because the spermatozoon is a highly specialized cell that expresses a diverse array of biological properties to achieve fertilization [3]. Additionally, in at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results [1]. When investigations of the female partner are also normal, the couple are classified as unexplained or idiopathic [4]. There is ongoing research into the development of new methods to complement the conventional semen analysis routinely used to assess infertility. One endpoint of interest is sperm DNA integrity, which can be assessed by a variety of assays, such as the sperm chromatin structure assay (SCSA) [5,6], the sperm DNA integrity assay (SDIa) [7,8], the terminal deoxynucleotidyl-transferase-mediated dUDP nick-end labeling assay [9], the in situ nick translation (ISNT) assay [10], and the single cell microgel electrophoresis (COMET) assay [11]. Methods focusing on the characterization of sperm chromatin condensation and stability are able to reveal hidden anomalies in the structural organization of sperm DNA and have been receiving growing attention. In particular, flow cytometric approaches allow rapid measurements to be carried out with a high level of precision, objectivity, and consistency with reliable results [12]. Both the SCSA and SDIa are flow cytometric methods that may identify spermatozoa with abnormal chromatin packaging, which could be the result of DNA breaks and/or derailments in protamine quantity and composition and/or an insufficient level of disulfide groups [6–8,13]. Acridine orange staining is used, after a low pH challenge, to distinguish between native DNA and fragmented DNA. Furthermore, in various studies, men with sperm DNA fragmentation (>27–30%) were placed into a statistical group taking a longer time to pregnancy, more in vitro fertilization (IVF) cycles, increased miscarriages or no pregnancy [7,14–17]. The objective of this study was to evaluate levels of sperm nuclear DNA fragmentation in a population of infertile men with normal
standard semen parameters and to compare the results with those from men who had abnormal semen parameters, as well as with a control group of men with proven fertility.
Subjects, materials and methods Study population All infertile couples included in this study had had at least 1 year of regular unprotected intercourse with no pregnancy achieved. A medical history was obtained from the infertile patients, and an experienced urologist performed a genital examination. Study participants were infertile men with normal standard semen parameters on repeated analyses and infertile men with an abnormality in one or more of the standard semen parameters on repeated analyses. The female partners of these men had no history of untreated female-factor infertility and had a normal reproductive and sexual history as well as normal investigations (e.g. pelvic ultrasound, endometrial biopsy, and hysterosalpingogram). Infertile men with azoospermia or couples with untreated femalefactor infertility were excluded from the study. A group of healthy donors (n = 30) with proven fertility (i.e. fathered a child within the last 12 months) and with normal genital examinations were included as controls.
Semen samples Semen samples were obtained from infertile men (n = 202) and from fertile donors (n = 30) during March 2007 through April 2008, after informed consent. All samples were collected by masturbation, after a period of 3–5 days of sexual abstinence. After 30 min from the time of ejaculation (for semen liquefaction at room temperature), 500 ml of the raw semen sample was frozen into a LN2 tank at 196 8C for later SDIa.
Standard semen analysis Following liquefaction, semen specimens were evaluated for semen volume, appearance, pH, and viscosity. Manual semen analysis was performed according to World Health Organization (WHO) guidelines [1] to determine sperm concentration and motility. Samples (5 ml) of liquefied semen was loaded on a Cell-VU count-
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Original article ing chamber (Millennum Sciences, Inc., NY, USA) and examined under x200 magnification. Sperm concentration was expressed as 1 106 /ml semen, while motility was expressed as a percentage. Smears of the raw semen were stained using the Spermac stain kit (FertiPro N.V., Belgium) for assessment of sperm morphology using the WHO classification [1]. Immediately after staining, the smears were rinsed in distilled water, air-dried, and scored. In this study, normal values were: sperm concentration 20 106 /ml semen, motility 50% and normal sperm forms 14% [1].
Sperm DNA Integrity assay Sperm DNA fragmentation was determined using the acridine orange staining method on flow cytometry [7]. On the day of analysis, the samples were thawed and the test was performed as soon as possible thereafter. All buffers and samples were kept at 4–6oC and the test was run in a box with ice. After washing with TNE (0.15 M NaCl, 0.01 M TrisHCL, 1 mM EDTA, pH 7.4) buffer, the sperm concentration was adjusted to approximately 1–2 x 106 cells/ ml and 200 ml was placed in 12 x 75 flow cytometer tubes. Low-pH (pH 1.2) detergent solution (400 ml) containing 0.1% Triton X100, 0.15 M NaCl and 0.08 M HCl was added for 30 s, and was immediately followed by 1.2 ml staining buffer (phosphate-citrate buffer, pH 6.0) with 6 mg/l acridine orange (AO) (chromatographically purified; cat. 318337-1G, Sigma-Aldrich, USA). Cells were acquired on a FACScan flow cytometer (Becton Dickinson). A total of 5000 events were accumulated for each measurement at a low flow rate. 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 flow cytometric measurements of the metachromatic shift from green (native, double-stranded DNA) to red (denatured, single-stranded DNA) fluorescence and displayed as red (fragmented DNA) versus green (DNA stainability) fluorescence intensity cytogram patterns. Data for each acquired specimen was saved and analyzed using FlowJo software (TreeStar,Inc., Ashland, OR, USA). Computer gates were used to determine the proportion of spermatozoa with
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increased levels of red fluorescence (fragmented single-stranded DNA) and green fluorescence (native double-stranded DNA). The results were presented as a percentage of the sperm DNA fragmentation index (DFI).
Statistical analysis Comparisons between donors, infertile men with normal semen parameters, and infertile men with at least one abnormal semen parameter were performed using the Mann-Whitney test for continuous variables. Spearman correlation coefficients were used to evaluate relationships between continuous variables. Summary statistics were presented as median and inter-quartile values (25th and 75th percentiles). All hypothesis testing was two-sided, and a probability value of p < 0.05 was deemed significant. Calculations were performed using SPSS version 13.0 software for Windows.
Results Among the fertile donor group, the highest observed DFI was 29.97%; these cut-off values were used to classify infertile men into high (>30% DFI), and low (30% DFI) DNA damage groups. The distribution of 30 fertile donors and 202 infertile men, according to the results of standard semen analyses [1] and the SDIadefined sperm DNA fragmentation index (DFI), is illustrated in Fig. 1. Of the 30 fertile donors, only 1 (3.3%) had abnormal sperm concentration (14 106/ml) and motility (36%), while in the infertile group, 154 out of 202 patients (76.2%) had abnormalities in one or more of the standard sperm parameters. The remaining 48 patients (23.8%) were normal according to the WHO standards. However, levels of sperm DNA fragmentation, as measured by the DFI, of more than 30% were observed in 19 out of these 48 (39.6%) infertile men with otherwise normal standard sperm parameters, compared with 91 out of 154 (59.1%) infertile men with abnormal sperm parameters. A comparison of standard semen parameters and DFI in fertile donors and in infertile men with normal and abnormal standard semen parameters is shown in Table 1. The only significant difference between fertile donors and infertile men with normal
Original article
Figure 1
The distribution of 30 fertile donors and 202 infertile men, according to the results of standard semen analysis and the sperm DNA integrity assay (SDIa)-defined DNA fragmentation index (DFI), is illustrated. The term abnormal semen analysis is based on the finding of at least one parameter below the WHO thresholds (sperm concentration <20 106/ml, motility <50%, normal sperm forms <14%). The SDIa results were stratified by determining the upper limits of the DFI in the fertile donor group (30%). SDIa results in the infertile group were expressed as DFI >30% or 30%.
Table 1 Age, standard sperm parameters and SDIa-defined DNA fragmentation index (DFI) in fertile donors, infertile men with normal semen parameters, and infertile men with abnormal semen parameters Variable
Age Concentration (106/ml) Motility (%) Morphology (%) DFI (%)
Fertile
Infertile men with
Infertile men with
p value
donors
normal semen
abnormal semen
(n = 30)
parameters (n = 48)
parameters (n = 154)
A
B
C
34 (31, 37) 63 (51, 84)
35 (31, 37) 72 (46, 94)
35 (31, 40) 31 (18, 59)
0.85 0.88
0.87 0.0001
0.81 0.0001
60 (50, 68) 30 (20, 33) 12 (8, 15)
55 (51, 61) 23 (16, 32) 21 (11, 35)
29 (16, 41) 22 (7, 22) 26 (15, 42)
0.34 0.07 0.03
0.0001 0.0001 0.0001
0.0001 0.0001 0.10
SDIa, sperm DNA integrity assay; DFI, DNA fragmentation index.Values are median (25th and 75th percentiles).A = p value of fertile donors versus infertile men with normal semen parameters; B = p value of fertile donors versus infertile men with abnormal semen parameters; C = p value of infertile men with normal semen parameters versus infertile men with abnormal semen parameters.Mann-Whitney test was used for comparison and statistical significance was assessed at p < 0.05.
standard semen parameters was observed in the levels of DNA fragmentation (DFI: p = 0.03). However, no significant difference in DFI was observed between infertile men with normal and abnormal semen parameters (p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperms from infertile men with abnormal and normal semen parameters, but not in fertile donors (r = 0.26, p = 0.001 and r = 0.48, p = 0.0001, respectively). The median age, as well as the 25th and 75th percentile values, for infertile men with abnormal semen parameters were not significantly different from infertile men with normal
semen parameters (p = 0.81) or fertile men (p = 0.87) (Table 1).
Discussion The results from this study indicate that levels of SDIa-defined DNA fragmentation in sperm from infertile men with abnormal standard sperm parameters (i.e. concentration, motility, and normal forms) were significantly higher than the levels from fertile men. This observation is in agreement with the reports of several recent studies, which indicated a negative correlation between the percentage of spermato-
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Original article zoa with DNA damage and standard sperm parameters [16,18,19]. In view of the association between poor semen quality and increased sperm DNA damage, it is probable that spermatozoa selected for assisted reproductive technologies (ART) may originate from samples with high percentages of sperm with damaged DNA [17,20]. Since the initial reports of pregnancies using intracytoplasmic sperm injection (ICSI), tremendous progress has occurred in the treatment of male-factor infertility, apparently regardless of the sperm defect. Because a spermatozoon is injected directly into the cytoplasm of the mature oocyte during ICSI, classical sperm parameters or sperm–oocyte interactions are no longer relevant, which places increased emphasis on the quality of the sperm chromatin. It is unclear if ART, including ICSI, are effective in compensating for poor chromatin packaging and/or DNA damage, or if suboptimal chromatin integrity is responsible for the poor implantation rate (<20%) in the majority of ART patients. ICSI is the technique used primarily for the treatment of infertile men with poor sperm quality, thus a major concern would be the use of DNAdamaged spermatozoa to fertilize the oocyte, which may have adverse consequences such as fertilization failure, early embryo death, spontaneous abortion, childhood cancer, and infertility in the offspring. Lopes et al. have shown that men with sperm DNA damage of more than 25% are more likely to experience a fertilization rate of less than 20% after ICSI [21]. There is a substantial risk that spermatozoa carrying damaged DNA are being used for ART. The results of our study indicate that levels of sperm DNA fragmentation in infertile men with normal standard sperm parameters were significantly higher in comparison to the levels in the fertile group and were not significantly different from the levels in infertile men with abnormal standard sperm parameters. This finding has important diagnostic and prognostic value in the management of male infertility. Increased DNA damage may be, at least in part, responsible for low fertility in men who otherwise have normal standard semen parameters on repeated analyses and, as a result, are diagnosed as unexplained or idiopathic. Potential mechanisms of DNA damage include (1) defective chromatin condensation during spermatogenesis [22,23], (2) initiation
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of apoptosis during spermatogenesis or during transport of sperm through male or female genital tracts [18,23], and (3) oxidative stress by reactive oxygen species produced internally or externally [23,24]. These three mechanisms might independently or codependently be responsible for sperm DNA damage. The first mechanism postulates that sperm do 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 [22,23]. Furthermore, many investigators have correlated the presence of DNA damage with poor chromatin packaging. The second hypothesized mechanism relates to apoptotic DNA cleavage in germ cells [18,23]. Sperm DNA damage might result from an ‘abortive apoptosis’, which occurs in the later stages of germ cell development, resulting in sperm cells that are dying 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 post-testicular sites [23–25]. Our data on the associations between the sperm quality (sperm motility and morphology) and sperm DNA fragmentation are consistent with those of Zini et al. [26] and Giwercman et al. [27], who reported significant negative correlations between sperm DNA fragmentation (assessed by SCSA) and sperm quality. Irvine et al. also demonstrated a negative correlation between sperm DNA integrity (assessed by COMET and ISNT assays) and sperm quality [10]. The association between DFI and motility indicates that normally motile sperm may or may not have an intact DNA molecule. Based on the results of this study, it is possible that sperm DNA fragmentation analysis may be better at discriminating between infertile men and fertile men than standard semen analysis. Other recent independent studies have also suggested that sperm DNA fragmentation analysis is an independent test of sperm quality that may have better diagnostic capabilities than standard sperm parameters [14,28]. In conclusion, infertile men with normal standard semen parameters on repeated analysis should be evaluated for sperm DNA fragmentation. Sperm from infertile men with
Original article normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to the levels in infertile men with abnormal sperm parameters. Therefore,
this test may reveal a hidden abnormality of sperm nuclear DNA in those infertile men classified as idiopathic on the basis of apparently normal standard sperm parameters.
References [1] World Health Organization, Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. 4th edition. Cambridge, UK: Cambridge University Press; 1999. [2] Rowe PJ, Comhaire FH, Hargreave TB, Mellows HJ. WHO Manual for the Standardized Investigation and Diagnosis of the Infertile Couple. Cambridge, UK: Cambridge University Press; 1993. [3] Holt WV, Van Look KJW. Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality. Reproduction 2004;127:527–35. [4] Nieschlag E. Scope and goal of andrology. In: Nieschlag E, Behre HM, editors. Andrology: Male Reproductive Health and Dysfunction. Berlin: Springer; 2000. p. 1–8. [5] Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, et al. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999;14:1039–49. [6] Evenson DP, Jost LK. Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci 2000;22:169–89. [7] Coulam CB, Ahlering P, Rodriquez H, Keskintepe L, Sher G, Roussev R. Pregnancy outcome after IVF/ET among couples who have elevated DNA fragmentation determined by the sperm DNA integrity assay (SDIa). Fert Steril 2003;80(Suppl. 3):207 (Abstract). [8] Ahlering P, Keskintepe L, Sher G, Fisch JD, Danner L, Maasarani G. The sperm DNA integrity assay (SDI(TM)) a valuable method for assessing sperm vitality in preparation for in vitro fertilization (IVF). Fert Steril 2003;80(Suppl 3):113 (Abstract). [9] Host E, Lindenberg S, Smidt-Jensen S. DNA strand breaks in human spermatozoa: correlation with fertilization in vitro in oligozoospermic men and in men with unexplained infertility. Acta Obstet Gynecol Scand 2000;79:189–93.
[10] Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl 2000;21:33–44. [11] Trisini AT, Singh NP, Duty SM, Hauser R. Relationship between human semen parameters and deoxyribonucleic acid damage assessed by the neutral comet assay. Fertil Steril 2004;82:1623–32. [12] Cordelli E, Eleuteri P, Leter G, Rescia M, Spano` M. Flow cytometry applications in the evaluation of sperm quality: semen analysis, sperm function and DNA integrity. Contraception 2005;72:273–9. [13] Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril 2000;73:43–50. [14] Larson KL, De Jonge CJ, Barnes AM, Jost LK, Evenson DP. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod 2000;15:1717–22. [15] Larson-Cook KL, Brannian JD, Hansen KA, Kasperson KM, Aarnold ET, Evenson DP. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertil Steril 2003;80:895–902. [16] Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, et al. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003;79:1597–605. [17] Roussev RG, Coulam CB. Elevated sperm DNA fragmentation is associated with reproductive failure. Am J Reprod Immunol 2004;51:472. [18] Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi P, Bianchi U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 1999;4:31.
[19] Saleh RA, Agarwal A, Nelson DR, Nada EA, El Tonsy MH, Alvares JG, et al. Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertil Steril 2002;78:313–8. [20] Bungum M, Humaidan P, Spano M, Jepson K, Bungum L, Giwercman A. The predictive value of sperm chromatin structure assay (SCSA) parameters for the outcome of intrauterine insemination, IVF and ICSI. Hum Reprod 2004;19:1401–8. [21] Lopes S, Sun J, Jurisicova A, Meriano J, Casper RF. Semen deoxyribonucleic acid fragmentation is increased in poor quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998;69:528–32. [22] Zini A, Libman J. Sperm DNA damage: clinical significance in the era of assisted reproduction. CMAJ 2006;175:495–500. [23] O’Brien J, Zini A. Sperm DNA integrity and male infertility. Urology 2005;65: 16–22. [24] Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001;122:497–506. [25] Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 2000;15:1338–44. [26] Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril 2001;75:674–7. [27] Giwercman A, Richthoff J, Hjollund H, Bonde JP. Correlation between sperm motility and sperm chromatin structure assay parameters. Fertil Steril 2003;80: 1404–12. [28] Zini A, Kamal K, Phang D, Willis J, Jarvi K. Biologic variability of sperm DNA denaturation in infertile men. Urology 2001;58: 258–61.
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Keywords Sperm DNA fragmentation Flow cytometry Male infertility Sperm
Tzvetan H. Lukanov, MD, PhD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, and Center for Reproductive Health, Medical University of Pleven 1, Pleven, Bulgaria Danail I. Lichev, MD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, and Center for Reproductive Health, Medical University of Pleven, Pleven, Bulgaria Emiliana I. Konova, MD, PhD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, and Center for Reproductive Health, Medical University of Pleven, Pleven, Bulgaria Alkan I. Emin, MD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, and Center for Reproductive Health, Medical University of Pleven, Pleven, Bulgaria Nina P. Ayvazova Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, Bulgaria Anjelika V. Velkova, MD, PhD Department of Social Medicine, Medical University of Pleven, Pleven, Bulgaria Roumen G. Roussev, MD, PhD Medical Center ‘Clinical Institute for Reproductive Medicine’, Pleven, Bulgaria and Millenova Immunology Laboratories, Chicago, Illinois, USA
Flow cytometric measurement of sperm nuclear DNA fragmentation in infertile men with normal standard sperm parameters Tzvetan H. Lukanov, Danail I. Lichev, Emiliana I. Konova, Alkan I. Emin, Nina P. Ayvazova, Anjelika V. Velkova and Roumen G. Roussev Abstract Background: Male-factor infertility plays a role in approximately 50% of infertile couples. In at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results. When diagnostic work-up of the female partner is also normal, they are classified as idiopathic. The objective of this study was to evaluate the levels of sperm nuclear DNA fragmentation in a population of infertile men with normal standard semen parameters and to compare their results with those from men who had abnormal semen parameters, as well as with a control group of fertile men. Methods: Semen samples were obtained from 202 infertile men and 30 fertile donors. Standard semen analysis was performed according to the World Health Organization guidelines. Flow cytometry has been extensively used to study sperm DNA fragmentation and the results are expressed as the percentage of sperm DNA fragmentation index (DFI). Results: Of the 202 patients, 48 (23.8%) had normal standard sperm parameters, while 154 (76.2%) had an abnormality in one or more of these parameters. DFI in infertile men with normal sperm parameters was significantly higher than in fertile donors (p = 0.03), but not significantly different from infertile men with abnormal sperm parameters (p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperm from infertile men with abnormal and normal semen parameters, but not in fertile donors (r = 0.26, p = 0.001 and r = 0.48, p = 0.0001, respectively). Conclusion: Sperm from infertile men with normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to levels in infertile men with abnormal sperm parameters. Sperm DNA fragmentation analysis is an independent test of sperm quality and has an important diagnostic value in the evaluation of male infertility. ß 2008 WPMH GmbH. Published by Elsevier Ireland Ltd.
Introduction
E-mail: [email protected]
Online 20 November 2008
50
Male-factor infertility plays a role in approximately 50% of infertile couples [1]. Standard
Vol. 6, No. 1, pp. 50–55, March 2009
semen analysis consists of measuring a variety of semen parameters, including volume, pH, sperm concentration, vitality, and morphology, the presence of leukocytes and immature
ß 2008 WPMH GmbH. Published by Elsevier Ireland Ltd.
Original article germ cells, and sperm motility [2]. 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, diagnosing defective sperm function by conventional semen analysis is difficult because the spermatozoon is a highly specialized cell that expresses a diverse array of biological properties to achieve fertilization [3]. Additionally, in at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results [1]. When investigations of the female partner are also normal, the couple are classified as unexplained or idiopathic [4]. There is ongoing research into the development of new methods to complement the conventional semen analysis routinely used to assess infertility. One endpoint of interest is sperm DNA integrity, which can be assessed by a variety of assays, such as the sperm chromatin structure assay (SCSA) [5,6], the sperm DNA integrity assay (SDIa) [7,8], the terminal deoxynucleotidyl-transferase-mediated dUDP nick-end labeling assay [9], the in situ nick translation (ISNT) assay [10], and the single cell microgel electrophoresis (COMET) assay [11]. Methods focusing on the characterization of sperm chromatin condensation and stability are able to reveal hidden anomalies in the structural organization of sperm DNA and have been receiving growing attention. In particular, flow cytometric approaches allow rapid measurements to be carried out with a high level of precision, objectivity, and consistency with reliable results [12]. Both the SCSA and SDIa are flow cytometric methods that may identify spermatozoa with abnormal chromatin packaging, which could be the result of DNA breaks and/or derailments in protamine quantity and composition and/or an insufficient level of disulfide groups [6–8,13]. Acridine orange staining is used, after a low pH challenge, to distinguish between native DNA and fragmented DNA. Furthermore, in various studies, men with sperm DNA fragmentation (>27–30%) were placed into a statistical group taking a longer time to pregnancy, more in vitro fertilization (IVF) cycles, increased miscarriages or no pregnancy [7,14–17]. The objective of this study was to evaluate levels of sperm nuclear DNA fragmentation in a population of infertile men with normal
standard semen parameters and to compare the results with those from men who had abnormal semen parameters, as well as with a control group of men with proven fertility.
Subjects, materials and methods Study population All infertile couples included in this study had had at least 1 year of regular unprotected intercourse with no pregnancy achieved. A medical history was obtained from the infertile patients, and an experienced urologist performed a genital examination. Study participants were infertile men with normal standard semen parameters on repeated analyses and infertile men with an abnormality in one or more of the standard semen parameters on repeated analyses. The female partners of these men had no history of untreated female-factor infertility and had a normal reproductive and sexual history as well as normal investigations (e.g. pelvic ultrasound, endometrial biopsy, and hysterosalpingogram). Infertile men with azoospermia or couples with untreated femalefactor infertility were excluded from the study. A group of healthy donors (n = 30) with proven fertility (i.e. fathered a child within the last 12 months) and with normal genital examinations were included as controls.
Semen samples Semen samples were obtained from infertile men (n = 202) and from fertile donors (n = 30) during March 2007 through April 2008, after informed consent. All samples were collected by masturbation, after a period of 3–5 days of sexual abstinence. After 30 min from the time of ejaculation (for semen liquefaction at room temperature), 500 ml of the raw semen sample was frozen into a LN2 tank at 196 8C for later SDIa.
Standard semen analysis Following liquefaction, semen specimens were evaluated for semen volume, appearance, pH, and viscosity. Manual semen analysis was performed according to World Health Organization (WHO) guidelines [1] to determine sperm concentration and motility. Samples (5 ml) of liquefied semen was loaded on a Cell-VU count-
Vol. 6, No. 1, pp. 50–55, March 2009
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Original article ing chamber (Millennum Sciences, Inc., NY, USA) and examined under x200 magnification. Sperm concentration was expressed as 1 106 /ml semen, while motility was expressed as a percentage. Smears of the raw semen were stained using the Spermac stain kit (FertiPro N.V., Belgium) for assessment of sperm morphology using the WHO classification [1]. Immediately after staining, the smears were rinsed in distilled water, air-dried, and scored. In this study, normal values were: sperm concentration 20 106 /ml semen, motility 50% and normal sperm forms 14% [1].
Sperm DNA Integrity assay Sperm DNA fragmentation was determined using the acridine orange staining method on flow cytometry [7]. On the day of analysis, the samples were thawed and the test was performed as soon as possible thereafter. All buffers and samples were kept at 4–6oC and the test was run in a box with ice. After washing with TNE (0.15 M NaCl, 0.01 M TrisHCL, 1 mM EDTA, pH 7.4) buffer, the sperm concentration was adjusted to approximately 1–2 x 106 cells/ ml and 200 ml was placed in 12 x 75 flow cytometer tubes. Low-pH (pH 1.2) detergent solution (400 ml) containing 0.1% Triton X100, 0.15 M NaCl and 0.08 M HCl was added for 30 s, and was immediately followed by 1.2 ml staining buffer (phosphate-citrate buffer, pH 6.0) with 6 mg/l acridine orange (AO) (chromatographically purified; cat. 318337-1G, Sigma-Aldrich, USA). Cells were acquired on a FACScan flow cytometer (Becton Dickinson). A total of 5000 events were accumulated for each measurement at a low flow rate. 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 flow cytometric measurements of the metachromatic shift from green (native, double-stranded DNA) to red (denatured, single-stranded DNA) fluorescence and displayed as red (fragmented DNA) versus green (DNA stainability) fluorescence intensity cytogram patterns. Data for each acquired specimen was saved and analyzed using FlowJo software (TreeStar,Inc., Ashland, OR, USA). Computer gates were used to determine the proportion of spermatozoa with
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increased levels of red fluorescence (fragmented single-stranded DNA) and green fluorescence (native double-stranded DNA). The results were presented as a percentage of the sperm DNA fragmentation index (DFI).
Statistical analysis Comparisons between donors, infertile men with normal semen parameters, and infertile men with at least one abnormal semen parameter were performed using the Mann-Whitney test for continuous variables. Spearman correlation coefficients were used to evaluate relationships between continuous variables. Summary statistics were presented as median and inter-quartile values (25th and 75th percentiles). All hypothesis testing was two-sided, and a probability value of p < 0.05 was deemed significant. Calculations were performed using SPSS version 13.0 software for Windows.
Results Among the fertile donor group, the highest observed DFI was 29.97%; these cut-off values were used to classify infertile men into high (>30% DFI), and low (30% DFI) DNA damage groups. The distribution of 30 fertile donors and 202 infertile men, according to the results of standard semen analyses [1] and the SDIadefined sperm DNA fragmentation index (DFI), is illustrated in Fig. 1. Of the 30 fertile donors, only 1 (3.3%) had abnormal sperm concentration (14 106/ml) and motility (36%), while in the infertile group, 154 out of 202 patients (76.2%) had abnormalities in one or more of the standard sperm parameters. The remaining 48 patients (23.8%) were normal according to the WHO standards. However, levels of sperm DNA fragmentation, as measured by the DFI, of more than 30% were observed in 19 out of these 48 (39.6%) infertile men with otherwise normal standard sperm parameters, compared with 91 out of 154 (59.1%) infertile men with abnormal sperm parameters. A comparison of standard semen parameters and DFI in fertile donors and in infertile men with normal and abnormal standard semen parameters is shown in Table 1. The only significant difference between fertile donors and infertile men with normal
Original article
Figure 1
The distribution of 30 fertile donors and 202 infertile men, according to the results of standard semen analysis and the sperm DNA integrity assay (SDIa)-defined DNA fragmentation index (DFI), is illustrated. The term abnormal semen analysis is based on the finding of at least one parameter below the WHO thresholds (sperm concentration <20 106/ml, motility <50%, normal sperm forms <14%). The SDIa results were stratified by determining the upper limits of the DFI in the fertile donor group (30%). SDIa results in the infertile group were expressed as DFI >30% or 30%.
Table 1 Age, standard sperm parameters and SDIa-defined DNA fragmentation index (DFI) in fertile donors, infertile men with normal semen parameters, and infertile men with abnormal semen parameters Variable
Age Concentration (106/ml) Motility (%) Morphology (%) DFI (%)
Fertile
Infertile men with
Infertile men with
p value
donors
normal semen
abnormal semen
(n = 30)
parameters (n = 48)
parameters (n = 154)
A
B
C
34 (31, 37) 63 (51, 84)
35 (31, 37) 72 (46, 94)
35 (31, 40) 31 (18, 59)
0.85 0.88
0.87 0.0001
0.81 0.0001
60 (50, 68) 30 (20, 33) 12 (8, 15)
55 (51, 61) 23 (16, 32) 21 (11, 35)
29 (16, 41) 22 (7, 22) 26 (15, 42)
0.34 0.07 0.03
0.0001 0.0001 0.0001
0.0001 0.0001 0.10
SDIa, sperm DNA integrity assay; DFI, DNA fragmentation index.Values are median (25th and 75th percentiles).A = p value of fertile donors versus infertile men with normal semen parameters; B = p value of fertile donors versus infertile men with abnormal semen parameters; C = p value of infertile men with normal semen parameters versus infertile men with abnormal semen parameters.Mann-Whitney test was used for comparison and statistical significance was assessed at p < 0.05.
standard semen parameters was observed in the levels of DNA fragmentation (DFI: p = 0.03). However, no significant difference in DFI was observed between infertile men with normal and abnormal semen parameters (p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperms from infertile men with abnormal and normal semen parameters, but not in fertile donors (r = 0.26, p = 0.001 and r = 0.48, p = 0.0001, respectively). The median age, as well as the 25th and 75th percentile values, for infertile men with abnormal semen parameters were not significantly different from infertile men with normal
semen parameters (p = 0.81) or fertile men (p = 0.87) (Table 1).
Discussion The results from this study indicate that levels of SDIa-defined DNA fragmentation in sperm from infertile men with abnormal standard sperm parameters (i.e. concentration, motility, and normal forms) were significantly higher than the levels from fertile men. This observation is in agreement with the reports of several recent studies, which indicated a negative correlation between the percentage of spermato-
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Original article zoa with DNA damage and standard sperm parameters [16,18,19]. In view of the association between poor semen quality and increased sperm DNA damage, it is probable that spermatozoa selected for assisted reproductive technologies (ART) may originate from samples with high percentages of sperm with damaged DNA [17,20]. Since the initial reports of pregnancies using intracytoplasmic sperm injection (ICSI), tremendous progress has occurred in the treatment of male-factor infertility, apparently regardless of the sperm defect. Because a spermatozoon is injected directly into the cytoplasm of the mature oocyte during ICSI, classical sperm parameters or sperm–oocyte interactions are no longer relevant, which places increased emphasis on the quality of the sperm chromatin. It is unclear if ART, including ICSI, are effective in compensating for poor chromatin packaging and/or DNA damage, or if suboptimal chromatin integrity is responsible for the poor implantation rate (<20%) in the majority of ART patients. ICSI is the technique used primarily for the treatment of infertile men with poor sperm quality, thus a major concern would be the use of DNAdamaged spermatozoa to fertilize the oocyte, which may have adverse consequences such as fertilization failure, early embryo death, spontaneous abortion, childhood cancer, and infertility in the offspring. Lopes et al. have shown that men with sperm DNA damage of more than 25% are more likely to experience a fertilization rate of less than 20% after ICSI [21]. There is a substantial risk that spermatozoa carrying damaged DNA are being used for ART. The results of our study indicate that levels of sperm DNA fragmentation in infertile men with normal standard sperm parameters were significantly higher in comparison to the levels in the fertile group and were not significantly different from the levels in infertile men with abnormal standard sperm parameters. This finding has important diagnostic and prognostic value in the management of male infertility. Increased DNA damage may be, at least in part, responsible for low fertility in men who otherwise have normal standard semen parameters on repeated analyses and, as a result, are diagnosed as unexplained or idiopathic. Potential mechanisms of DNA damage include (1) defective chromatin condensation during spermatogenesis [22,23], (2) initiation
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of apoptosis during spermatogenesis or during transport of sperm through male or female genital tracts [18,23], and (3) oxidative stress by reactive oxygen species produced internally or externally [23,24]. These three mechanisms might independently or codependently be responsible for sperm DNA damage. The first mechanism postulates that sperm do 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 [22,23]. Furthermore, many investigators have correlated the presence of DNA damage with poor chromatin packaging. The second hypothesized mechanism relates to apoptotic DNA cleavage in germ cells [18,23]. Sperm DNA damage might result from an ‘abortive apoptosis’, which occurs in the later stages of germ cell development, resulting in sperm cells that are dying 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 post-testicular sites [23–25]. Our data on the associations between the sperm quality (sperm motility and morphology) and sperm DNA fragmentation are consistent with those of Zini et al. [26] and Giwercman et al. [27], who reported significant negative correlations between sperm DNA fragmentation (assessed by SCSA) and sperm quality. Irvine et al. also demonstrated a negative correlation between sperm DNA integrity (assessed by COMET and ISNT assays) and sperm quality [10]. The association between DFI and motility indicates that normally motile sperm may or may not have an intact DNA molecule. Based on the results of this study, it is possible that sperm DNA fragmentation analysis may be better at discriminating between infertile men and fertile men than standard semen analysis. Other recent independent studies have also suggested that sperm DNA fragmentation analysis is an independent test of sperm quality that may have better diagnostic capabilities than standard sperm parameters [14,28]. In conclusion, infertile men with normal standard semen parameters on repeated analysis should be evaluated for sperm DNA fragmentation. Sperm from infertile men with
Original article normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to the levels in infertile men with abnormal sperm parameters. Therefore,
this test may reveal a hidden abnormality of sperm nuclear DNA in those infertile men classified as idiopathic on the basis of apparently normal standard sperm parameters.
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