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• W2078742078 abstract "Routine semen analysis, which includes assessment of sperm motility, viability, and morphology, does not always provide complete diagnostic information as men demonstrating standard scores on these specific parameters sometimes remain infertile. In infertile men who had been subgrouped on the basis of sperm count, motility, morphology, and presence of leukocytes, an evaluation of sperm function and reactive oxygen species (ROS) was found to be effective tools to detect sperm pathologies. Routine semen analysis, which includes assessment of sperm motility, viability, and morphology, does not always provide complete diagnostic information as men demonstrating standard scores on these specific parameters sometimes remain infertile. In infertile men who had been subgrouped on the basis of sperm count, motility, morphology, and presence of leukocytes, an evaluation of sperm function and reactive oxygen species (ROS) was found to be effective tools to detect sperm pathologies. In recent years, there has been a growing concern with the diagnostic accuracy of sperm-specific laboratory tests of male factor infertility, where defective sperm function is one of the most common causative factors (1Sikka S.C. Oxidative stress and role of antioxidants in normal and abnormal sperm function.Front Biosci. 1996; 1: e78-e86Crossref PubMed Scopus (319) Google Scholar, 2Schill W.-B. Hankel R. Advancement in biochemical assays in andrology.Asian J Androl. 1999; 1: 45-51PubMed Google Scholar). Because no single test can evaluate all the steps involved in fertilization, a combination of assays complimenting each other is preferred to provide a comprehensive evaluation of sperm function leading to fertilization. Sperm penetration into the oocyte is linked to two specific sperm attributes, which can be determined by tests for acrosome status and plasma membrane integrity. Sperm nuclear chromatin decondensation (NCD), essential for pronuclei formation before the final step of the male and female gamete nucleus fusion, has also been evaluated in vitro (3Gopalkrishnan K. Hinduja I.N. Kumar T.C. Invitro decondensation of nuclear chromatin of human spermatozoa: assessing fertilizing potential.Arch Androl. 1991; 27: 43-50Crossref PubMed Scopus (37) Google Scholar). Reduced test scores of sperm function on these aspects are reportedly associated with unsustainable embryo development leading to early pregnancy loss (4Saxena P. Misro M.M. Chaki S.P. Chopra K. Roy S. Nandan D. Is abnormal sperm function an indicator among couples with recurrent pregnancy loss?.Fertil Steril. 2008; 90: 1854-1858Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, reports have been limited on the diagnostic value of these tests in different categories of infertile men with various sperm pathologies. Oxidative stress in semen arises from an imbalance between reactive oxygen species (ROS) generation and the scavenging ability of seminal antioxidants (5Pasqualotto F.F. Sharma R.K. Kobayashi H. Nelson D.R. Thomas Jr., A.J. Agarwal A. Oxidative stress in normospermic men undergoing infertility evaluation.J Androl. 2001; 22: 316-322PubMed Google Scholar). Reactive oxygen species are highly reactive oxidizing agents belonging to the class of free radicals. Oxidative stress influencing male fertility has been well documented (6Agarwal A. Gupta S. Sikka S. The role of free radicals and antioxidants in reproduction.Curr Opin Obstet Gynecol. 2006; 18: 325-332Crossref PubMed Scopus (333) Google Scholar, 7Aitken R.J. Sperm function tests and fertility.Int J Androl. 2006; 29 (105–8): 69-75Crossref PubMed Scopus (214) Google Scholar, 8Agarwal A. Saleh R.A. Bedaiwy M.A. Role of reactive oxygen species in the pathophysiology of human reproduction.Fertil Steril. 2003; 79: 829-843Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar). The aerobic metabolism of human sperm results in the production of different ROS, which are potentially harmful not only to the sperm plasma membrane with its high content of polyunsaturated fatty acids but also to the core nuclear chromatin through high frequency single or double DNA strand breaks (9Saleh R.A. Agarwal A. Nelson D.R. Nada E.A. El-Tonsy M.H. Alvarez J.G. et al.Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study.Fertil Steril. 2002; 78: 313-318Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 10Erenpreiss J. Hlevicka S. Zalkalns J. Erenpreisa J. Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples.J Androl. 2002; 23: 717-723PubMed Google Scholar). However, the degree of ROS-induced adverse effects on sperm attributes depends on the nature, amount, and duration of exposure. Thus, assessment of oxidative stress in infertile men is essential, and can be monitored with the help of a variety of indicators using either sperm or seminal plasma. Measurement of the rate of ROS generation by luminol induced chemiluminescence has been the most common method of quantifying ROS from sperm (11Saleh R.A. Agarwal A. Oxidative stress and male infertility: from research bench to clinical practice.J Androl. 2002; 23: 737-752PubMed Google Scholar). On the other hand, a test for total antioxidant capacity (TAC) is used to determine if the seminal fluid has adequate scavenging ability (12Kolettis P. Sharma R.K. Pasqualotto F. Nelson D. Thomas Jr., A.J. Agarwal A. The effects of seminal oxidative stress on fertility after vasectomy reversal.Fertil Stertil. 1999; 71: 249-255Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In addition, enzymatic antioxidant status is evaluated by measurement of activities of super oxide dismutase (SOD) (13Murawski M. Saczko J. Marcinkowska A. Chwilkowska A. Grybos M. Banas T. Evaluation of superoxide dismutase activity and its impact on semen quality parameters of infertile men.Folia Histochem Cytobiol. 2007; 45: S123-S126PubMed Google Scholar) and catalase (14Khosrowbeygi A. Zarghami N. Levels of oxidative stress biomarkers in seminal plasma and their relationship with seminal parameters.BMC Clin Pathol. 2007; 7: 6-17Crossref PubMed Scopus (99) Google Scholar) present in the seminal plasma. As oxidative stress is expected to affect all aspects of sperm function, it is prudent to assume that measurable scores would provide a predictive assessment, but this is not entirely true as we have learned. We not only assessed all these parameters but also examined the relationship of each of these with selected sperm function scores in fertile men and in different subgroups of infertile men. The infertile men were subgrouped on the basis of normal and subnormal sperm motility to demonstrate that sperm function independent of motility is affected. Because neutrophils in the semen could be the source of production of ROS in addition to sperm (15Sharma R.K. Agarwal A. Role of reactive oxygen species in male infertility.Urology. 1996; 48: 835-850Abstract Full Text PDF PubMed Scopus (749) Google Scholar), men with leucocytospermia were grouped together and studied separately. No statistically significant difference in sperm function scores was observed between the proven fertile controls and the infertile men who had good sperm count and motility. Sperm viability in the other infertile subgroups showed a statistically significant decline (P<.001), except for the leucocytospermia group, which showed good sperm motility (Fig. 1A ). However, when analyzed with the hypo-osmotic swelling (HOS) test, the score decrease was statistically significant in all the infertile subgroups (see Fig. 1B). The pattern was identical when the semen samples were assessed for acrosome status (see Fig. 1C) and nuclear chromatin decondensation (see Fig. 1D). Assessment of oxidative stress indicated that there was no statistically significant difference in enzyme activities (SOD, catalase) or TAC observed between the proven fertile and the normospermic infertile groups. However, in the rest of the infertile subgroups these values were statistically significantly altered, barring a few exceptions. The sensitivity of the SOD assay (Fig. 2A ) comprehensively matched with the TAC estimation (see Fig. 2C), which displayed a decreasing trend among all infertile groups excepting the oligospermic subgroup. The pattern was similar when catalase (see Fig. 2B) was compared with TAC values with the sole exception of the leucocytospermia subgroup. In contrast, the ROS levels were found to be statistically significantly high in all infertile men including the normospermic subgroup (see Fig. 2D). The primary source of ROS production in the male reproductive tract comes either from sperm or infiltrating leucocytes (15Sharma R.K. Agarwal A. Role of reactive oxygen species in male infertility.Urology. 1996; 48: 835-850Abstract Full Text PDF PubMed Scopus (749) Google Scholar). The half-life of ROS is very short (16Lunec J. Oxygen radicals: their measurement in vivo.Anal Proc. 1989; 26: 130-131Google Scholar). Thus, the impact of contaminating leucocytes on sperm function would depend on the number of cells involved, their state of activation and levels of free radical generation, and their length of exposure to sperm cells. Therefore, semen with leukocyte infiltrations, besides those beyond a certain level, does not sometimes show a major impact on sperm function in vivo, albeit influencing fertilization rates in vitro (17Baker H.W. Brindle J. Irvine D.S. Aitken R.J. Protective effect of antioxidants on the impairment of sperm motility by activated poly-morphonuclear leucocytes.Fertil Steril. 1996; 65: 411-419Abstract Full Text PDF PubMed Google Scholar). In our studies, too, sperm motility and viability were not statistically significantly affected in one subgroup of leucocytospermic men. However, HOS, acrosome status, and NCD scores were found to be statistically significantly (P<.01) attenuated compared with proven fertile controls. In contrast, consistent with the reduction in sperm motility in the other subgroup (leukocytospermic men with poor motility), all the sperm function scores were found to be uniformly affected. Statistically significant trends in ROS levels have emerged in infertile men with leucocytospermia. Both leucocytospermia subgroups demonstrated the presence of statistically significantly (P<.001) higher levels of ROS in sperm compared with the proven fertile controls. However, between the two groups, ROS levels were much higher in those whose sperm motility was statistically significantly affected. For the first time, we have used the sperm function test to demonstrate altered sperm characteristics/function in leucocytospermic men who retain good sperm motility. The viability of sperm in these men was comparable with that of the proven fertile volunteers. Earlier studies reported that ROS scavenging enzyme activity can be induced and that the specificity and the extent of induction depended on the cell type or source of ROS (18de Lamirande E. Gagnon C. Reactive oxygen species and human spermatozoa: II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility.J Androl. 1992; 13: 379-386PubMed Google Scholar). Except catalase, the SOD activity and the TAC in the leucocytospermia with good motility subgroup demonstrated a statistically significant decline. The marginal increase in catalase activity could be a compensatory mechanism producing more ROS scavengers, activating catalase, or both, which may result in the maintenance of normal sperm motility in the leucocytospermia with good motility subgroup. This also could explain why catalase-like rather than SOD-like activity was found to be statistically significantly increased in ROS-producing semen samples, as previously reported elsewhere (19Zini A. de Lamirande E. Gagnon C. Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase and catalase–like activities in seminal plasma.Int J Androl. 1993; 16: 183-188Crossref PubMed Scopus (320) Google Scholar). Sperm function scores correlated with oxidative stress parameters revealed certain unique and interesting findings. We found that ROS was positively (r = 0.319) correlated with sperm function in proven fertile men but was negatively correlated in all the infertile men. However, the extent of negative correlation varied extensively between the subgroups. In the normospermic group, a weak negative correlation (r = –0.212) was observed. The correlation was strongly negative (r = –0.5 to –0.8) in the rest of the subgroups. The correlation of TAC with sperm function was positive and statistically significant in all infertile men except for the oligospermic subgroup, with whom it was not statistically significant. Moderate to strong correlation was also observed between sperm function and the activities of SOD and catalase in the plasma. Multivariate discriminant analysis also has been reported to demonstrate that a combination of sperm attributes, such as semen volume, sperm count, percentage of motile forms, in correct proportions, provides the best diagnostic profile for delineating fertile men from suspected infertile men. Each variable has been assigned a weight to provide an overall semen quality score to distinguish the two populations (20Nallella K. Sharma R.K. Aziz N. Agarwal A. Significance of sperm characteristics in the evaluation of male fertility.Feril Steril. 2006; : 629-634Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). However, a significant overlap of sperm characteristics in fertile and infertile men also has been observed. Similarly, we found that subnormal sperm count and motility correlated statistically significantly well with subnormal sperm function. The sole exception was the subgroup of leucocytospermia with normal sperm motility, which demonstrated not only altered sperm function scores but also high levels of ROS. There are conflicting reports about the usefulness of ROS measurement as a better predictor of male factor infertility. No significant relationship has been reported between ROS production and sperm motility (21Whittington K. Harrison S.C. Willliams K.M. Day J.L. McLaughlin E.A. Hull M.G. et al.Reactive oxygen species (ROS) production and the outcome of diagnostic test of sperm function.Int J Androl. 1996; 22: 236-242Crossref Scopus (56) Google Scholar). Identical findings also have been reported in patients with prostatitis in whom seminal oxidative stress was not associated with a decline in sperm count, motility, or morphology compared with normal controls (22Pasqualotto F.F. Sharma R.K. Nelson D.R. Thomas Jr., A.J. Agarwal A. Relationship between oxidative stress, semen characteristics and clinical diagnosis in men undergoing infertility investigation.Fertil Steril. 2000; 73: 459-464Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). However, in our study, high ROS levels were uniformly found in all the infertile subgroups, with teratospermia topping the chart (see Fig. 2D). Though sperm motility was unaffected in the three groups (i.e., normospermia, oligospermia, and leucocytospermia), statistically significantly high levels of ROS were measured in all the groups. Although ROS are necessary for successful reproductive processes such as sperm capacitation, motility, acrosome reaction, and oocyte fusion (23Garrido N. Meseguer M. Alvarez J. Simón C. Pellicer A. Remohí J. Relationship among standard semen parameters, glutathione peroxidase/glutathione reductase activity, and mRNA expression and reduced glutathione content in ejaculated spermatozoa from fertile and infertile men.Fertil Steril. 2004; 82: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24Aitken R.J. Paterson M. Fisher H. Buckingham D.W. van Duin M. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function.J Cell Sci. 1995; 8: 2017-2025Google Scholar), this is true only when their level remains within physiologic limits. In infertile men, ROS levels rise beyond physiologic limits; even normospermic men show a 1.23-fold increase in ROS levels when compared with proven fertile men. Compared with proven fertile men, normospermic men showed no statistically significant alteration in TAC levels. This implies that these men are not deficient of antioxidants (25Zini A. Garrels K. Phang D. Antioxidant activity in the semen of fertile and infertile men.Urology. 2000; 55: 922-926Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Thus, the oxidative stress could be related to an increase in ROS or ineffective use of antioxidants (26Meucci E. Milardi D. Mordente A. Martorana G.E. Giacchi E. De Marinis L. Mancini A. Total antioxidant capacity in patients with varicoceles.Fertil Steril. 2003; 79: 1577-1583Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Our findings indicate that sperm function, as determined by HOS, acrosome status, and NCD assessment, correlates statistically significantly with the total antioxidant capacity and also with the antioxidant enzyme activities (SOD, catalase) present in the seminal plasma of the majority of infertile men. However, neither ROS nor sperm function alone was enough for an accurate diagnosis of sperm pathology in infertile men, particularly in cases of normospermia or leucocytospermia for which a combined assessment is absolutely necessary. For differentiating fertile from infertile men, the sensitivity of sperm function scoring or assays for oxidative stress in the semen have their own inherent limitations. Sperm function scores fall within the normal range in both proven fertile and normospermic infertile men. Similarly, high ROS levels in leucocytospermic men may not be entirely due to sperm anomalies, as interference from leucocytes cannot be ruled out. On the other hand, although activities of SOD, catalase, or TAC in the seminal plasma correlate typically to altered sperm pathologies/sperm function test scores in various groups of infertile men, this has little diagnostic value with respect to the infertile normospermic subgroup." @default.
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• W2078742078 title "Sperm function and seminal oxidative stress as tools to identify sperm pathologies in infertile men" @default.
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