Ramya T, M.Sc., Man Mohan Misro, Ph.D., Devabrata Sinha, M.B., B.S., Deoki Nandan, M.D., and Sandeep Mithal, M.S.

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1 Altered levels of seminal nitric oxide, nitric oxide synthase, and enzymatic antioxidants and their association with sperm function in infertile subjects Ramya T, M.Sc., Man Mohan Misro, Ph.D., Devabrata Sinha, M.B., B.S., Deoki Nandan, M.D., and Sandeep Mithal, M.S. Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi, India Objective: To investigate the association between specific reactive nitrogen species and sperm function among infertile subjects. Design: Controlled trial. Setting: Fertility clinic at the National Institute of Health and Family Welfare, New Delhi, India. Patient(s): Proven fertile volunteers and subjects attending the above clinic. Intervention(s): Tests for hypo-osmotic swelling (HOS), nuclear chromatin decondensation (NCD), acrosomal status (AS) were performed, and total nitric oxide (NO), nitric oxide synthase (NOS), superoxide dismutase (SOD), and catalase were assayed in infertile subjects subgrouped on the basis of routine sperm parameters. Main Outcome Measure(s): Correlation between sperm function and total NO, SOD, catalase, and NOS levels in semen. Result(s): Most of the infertile subjects were found to possess high NO and NOS activity in the semen. Leukocytospermic subjects, despite having normal sperm motility and viability, demonstrated subnormal sperm function scores. Nitric oxide in semen was inversely associated with sperm function in normospermia, asthenospermia, leukocytospermia, leukocytoasthenospermia, and teratospermia. In oligospermia with normal or subnormal motility, NO was positively associated with sperm function. Attenuated activities of either SOD or catalase or both were observed in all infertile subjects except those with normospermia. Conclusion(s): Subnormal sperm function in normospermia, asthenospermia, and leukocytospermia may be due to the cytotoxic effect of increased NO, whereas in oligospermia the same may be due to low or suboptimal levels. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Sperm function, infertile subjects, semen analysis, reactive nitrogen species Nitric oxide (NO) is a free radical generated from the oxidation of L-arginine to L-citrulline by reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent nitric oxide synthase (NOS). Nitric oxide is diffusible, multifunctional, and acts as a transcellular messenger, being implicated in numerous physiologic and pathologic conditions (1). Nitric oxide, at physiologic concentrations, is relatively nonreactive, but most of its actions are mediated by activation of cyclic guanosine monophosphate production (2). It is reported that NO modulates sexual and reproductive functions in mammalian species (3, 4). The presence of NO in seminal plasma has been confirmed (5). The source of NO in seminal plasma may be either male reproductive organs or macrophages (6, 7). The production of NO in human and animal sperm has also been reported (8, 9). Received February 3, 2010; revised May 21, 2010; accepted June 16, 2010; published online August 4, R.T. has nothing to disclose. M.M.M. has nothing to disclose. D.S. has nothing to disclose. D.N. has nothing to disclose. S.M. has nothing to disclose. Supported by the National Institute of Health and Family Welfare, New Delhi, India. Presented at the Annual Congress of the Society of Andrology, December 20 22, 2009, Rajasthan, India. Reprint requests: Man Mohan Misro, Ph.D., Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Baba Gang Nath Marg, Munirka, New Delhi , India (FAX: ; mm_misro@yahoo.com). Nitric oxide is reported to be a novel mediator of sperm function (10). It has both positive and negative effects. The positive modulation reflects the role of NO in physiologic processes like sperm capacitation (11) and acrosome reaction (12). On the other hand, negative effects, which down-regulate sperm motility (13) and viability (14), probably represent the role of NO as an inflammatory mediator in response to chronic or subclinical infection. The available information thus supports the fact that although excessive NO concentrations are deleterious to sperms, low and controlled concentrations of NO are crucial in optimizing specific processes in sperm physiology. Defective sperm function has been recognized as one of the causes of male infertility. Mammalian sperm membranes, rich in polyunsaturated fatty acids, are prone to lipid peroxidation, which is the key mechanism of reactive oxygen species induced sperm damage leading to infertility (15, 16). Reactive oxygen species are highly reactive oxidizing agents belonging to a class of free radicals derived from oxygen, whereas reactive nitrogen species are damaging free radicals derived from nitrogen. Nitric oxide is one of the most potent free radicals of nitrogen. Besides sperm kinetics, other functional characteristics have never been assessed before with respect to possible deleterious effects of reactive nitrogen species in semen. The present study was, therefore, initiated to find the degree of correlation of nitrogen radicals with sperm function in various infertile groups. Because the presence of leukocytes in the semen may also affect sperm function, these subjects are grouped separately /$36.00 Fertility and Sterility â Vol. 95, No. 1, January doi: /j.fertnstert Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 FIGURE 1 Evaluation of sperm function test scores. There was no significant decline in sperm viability (A), hypo-osmotic swelling (HOS) (B), acrosomal status (AS) (C), and nuclear chromatin decondensation (NCD) (D) in normospermic (N) compared with proven-fertile (PF) subjects. However, in other infertile subjects (IF) the decrease was significant. The exception is leukocytospermia with good motility (L), in which the viability score was unaltered. A ¼ asthenospermia (normal sperm count with less motility); O ¼ oligospermia with good motility; OA ¼ oligospermia with poor motility; LA ¼ leukocytospermia with poor motility; T ¼ teratospermia. a P<.05; b P<.01; c P<.001. MATERIALS AND METHODS Subject Selection and Semen Analysis The study was conducted between December 2007 and November 2009 in the Fertility Clinic of the Department of Reproductive Biomedicine at the National Institute of Health and Family Welfare, New Delhi, India. Requisite clearance from the institutional review board was obtained. Subjects aged years attending the fertility clinic and having a minimum of 1 year of unprotected regular intercourse were included in the infertile group. The research was explained to them, and informed consent was obtained as per ethical guidelines. The infertile subjects (n ¼ 164) were further divided into seven subgroups: normospermia: normal sperm count and normal motility (n ¼ 23); asthenospermia: normal sperm count with low motility (n ¼ 28); oligospermia: subnormal sperm count ( /ml) and normal motility (n ¼ 14); oligoasthenospermia: both sperm count and motility subnormal (n ¼ 22); leukocytospermia: leukocytes in the semen (> /ml) with normal sperm motility (n ¼ 21); leukocytoasthenospermia: leukocytes in the semen (> /ml) with subnormal sperm motility (n ¼ 26); and teratospermia: morphologically abnormal sperm (n ¼ 10). After three confirmed observations of semen, the subjects were included in the respective categories. The female partners of these men had no history of untreated female factor infertility and presented a normal sexual history and investigation results (pelvic, ultrasound, hysterosalpingogram, and endometrial biopsy). Subjects with azoospermia, severe oligospermia (< /ml), complete loss of motility, or couples with untreated female factor infertility were excluded. Twenty male volunteers who had either fathered children or had an ongoing conceptus in the last 6 months were included as proven-fertile controls. Semen samples were collected by masturbation after 2 to 3 days of sexual abstinence in sterile plastic containers. After liquefaction, routine semen analyses (volume, ph, sperm count, motility, morphology, and presence of leukocytes) were carried out as per World Health Organization criteria (17). Sperm Viability by Dye Exclusion Sperm viability was studied as previously described (17). The number of stained cells was counted, and viability percentage was noted (normal: >75% of unstained cells). Hypo-osmotic Swelling Test A modified hypo-osmotic swelling test protocol developed in our laboratory as previously described (18) was adopted. The percentage of sperm with tail coiling was recorded (normal: >60% sperm with coiled tails). Acrosomal Status Test An acrosomal status test was carried out as previously described (19). The sperm were examined under a microscope, and percentage of sperm with halos was calculated (normal: >50%). Nuclear Chromatin Decondensation Test The nuclear chromatin decondensation test was performed as previously described (20). At the end of reaction, percentage of sperm with swollen chromatin was recorded (normal: >70% of decondensed sperms). 136 Ramya T et al. Seminal nitric oxide and sperm function Vol. 95, No. 1, January 2011

3 FIGURE 2 Levels of (A) total NO and activities of (B) NOS, (C) SOD, and (D) catalase in the semen. The NO in seminal plasma was directly proportional to NOS in sperm. Significantly high NO and NOS activity were observed in asthenospermia (A) and leukocytoasthenospermia (LA). On the other hand, oligoasthenospermia (OA) demonstrated a significant decrease in NO and NOS levels. With the exception of normospermia (N), antioxidant enzyme activities showed a decreasing trend in most of the infertile (IF) subjects when compared with proven-fertile (PF) controls. O ¼ oligospermia with good motility; L ¼ leukocytospermia with good motility; T ¼ teratospermia. a P<.05; b P<.01; c P<.001. Sperm Morphology and Leukocytes Sperm were examined microscopically after Papanicolaou staining (17) to record various abnormalities and presence of leukocytes. Semen showing <30% morphologically normal sperm was placed in the teratospermia group. Assay of Total NO in Seminal Plasma Total NO was measured as per the manufacturer s instructions using a commercially available kit (Stressgen; Assay Designs, Ann Arbor, MI). The kit involves the enzymatic conversion of nitrate to nitrite by the enzyme nitrate reductase, followed by colorimetric detection of nitrite. Assay for NOS Nitric oxide synthase activity was measured using a commercially available kit (Oxford Biomed Research, Oxford, MI). This kit uses an NADPH recycling system to allow NOS to operate linearly for hours as NO-derived nitrate and nitrite accumulate. The nitrate formed was converted to nitrite by the enzyme nitrate reductase. Nitrite was quantified using Griess Reagent. Superoxide Dismutase Assay Superoxide dismutase (SOD) in seminal plasma was assayed as previously described (21). Superoxide dismutase activity was expressed as units per milliliter. Catalase Assay Catalase was assayed by the method of Aebi (22). Using hydrogen peroxide (6 mm) as substrate, the activity was calculated and expressed as katals/milliliter. Statistical Analysis Two-tailed t test and analysis of variance were performed using SPSS statistical software (SPSS, Chicago, IL). Correlation coefficients were calculated by Pearson s two-tailed correlation analysis. A P value of <.05 was considered statistically significant. RESULTS Subnormal sperm function scores (Fig. 1) were characteristic of all infertile subjects except those with normospermia, who had scores comparable to those of proven-fertile subjects. In subjects with leukocytospermia and oligospermia, subnormal sperm function independent of sperm motility was observed. Sperm viability was affected along with sperm function in all the infertile subjects barring those with normospermia and leukocytospermia (Fig. 1). Nitric oxide levels in the seminal plasma were found to be directly proportional to NOS activity in sperm. Significantly high NOS activity (Fig. 2B) was seen in the infertile subjects with asthenospermia, leukocytospermia, and leukocytoasthenospermia. Nitric oxide synthase activity, but not NO concentration, was raised in leukocytospermia in which sperm motility was unaffected. No significant Fertility and Sterility â 137

4 FIGURE 3 Correlation analysis between seminal plasma NO concentration and sperm function score percentage. A significant positive correlation was observed in proven fertile subjects, whereas normospermia, teratospermia, and asthenospermia demonstrated a negative correlation. HOS ¼ hypo-osmotic swelling; NCD ¼ nuclear chromatin decondensation; AS ¼ acrosomal status. alteration in NO concentration (Fig. 2A) was found either in oligospermic or leukocytospermic subjects retaining normal motility. With the exception of normospermia, the activity of SOD or catalase or both was found to be markedly altered in all infertile groups (Fig. 2C and D). In leukocytospermia, a significant increase in catalase activity was observed. When seminal plasma NO was correlated with sperm function (Figs. 3 and 4), it presented a significant negative correlation in the asthenospermia, leukocytoasthenospermia, and leukocytospermia groups. The negative correlation was not significant (r ¼ 0.126) among normospermic subjects. Teratospermia showed poor association (r ¼ 0.084), whereas oligoasthenospermia and oligospermia showed a significant positive association (r ¼ 0.606, P<.001 and r ¼ 0.5, P<.001, respectively) of NO with sperm function. DISCUSSION Total NO levels in the seminal plasma corresponded well with NOS activity in sperm. In most of the infertile subjects (normospermia, asthenospermia, leukocytospermia, leukocytoasthenospermia) the semen presented high NO and NOS activity (Fig. 2A and B) compared with proven-fertile controls. The exceptions were the groups with low sperm count (oligospermia and oligoasthenospermia) and teratospermia. The increase in the levels of NO and NOS was statistically significant among those groups in which sperm motility was affected (asthenospermia, leukocytoasthenospermia). The decline in sperm motility might be attributed to the cytotoxic effects of excess reactive nitrogen species in the semen. The findings are in accordance with the reported observation that elevated NO levels inhibit sperm motion (23). Nitric oxide can reduce cellular adenosine triphosphate (ATP) levels by inhibiting the ATP-generating enzymatic activities of electron transport chain (24). The required energy for sperm motility comes from the mitochondria. Reduction in ATP sources/production results in insufficient energy levels, which affects sperm motility. In varicocele patients with reduced sperm motility, an inverse correlation between seminal plasma NO concentration and sperm motility was reported (25). Aberrant patterns of sperm endothelial NOS expression are also reportedly 138 Ramya T et al. Seminal nitric oxide and sperm function Vol. 95, No. 1, January 2011

5 FIGURE 4 Correlation analysis between seminal plasma NO levels and sperm function score percentage. The correlation was positive in oligoasthenospermia and oligospermia and was significantly negative in leukocytoasthenospermia and leukocytospermia. HOS ¼ hypoosmotic swelling; NCD ¼ nuclear chromatin decondensation; AS ¼ acrosomal status. associated with decreased sperm motility, possibly through the generation of excessive cytotoxic oxidants (26). In the oligospermia group there was a decline in seminal NO levels. This may be due to the presence of fewer sperm contributing to the nitrite/nitrate pool of the semen. There are contradictory reports on sperm concentration and seminal plasma nitrite levels. Revelli et al. (27) reported that nitrite levels in seminal plasma of normospermic men were comparable to those of mild to moderate oligospermic and even in severely oligo-/ azoospermic men. On the other hand, Battaglia et al. (28) demonstrtated significantly high seminal plasma nitrite/nitrate levels in normospermic vs. oligospermic men, which supports the present findings. In the leukocytospermia group, sperm motility and viability were not impaired despite the presence of leukocytes. Even though there was a significant increase in NOS activity, the NO levels were comparable to those of proven-fertile subjects. Both sperm and leukocytes, in such cases, may contribute to the total NOS activity. However, the presence of an endogenous NOS inhibitor has been reported to maintain the levels of NO at lower concentrations and thus prevent toxic damage to sperm (29). Nitric oxide synthase inhibitor in human seminal plasma has also been found by others (30). Although the presence of NOS inhibitor is not confirmed in the present study, the fact that sperm motility and viability remained unaffected in the leukocytospermia group suggests the possible existence of such an inhibitor. Attenuated activities of antioxidant enzymes, either SOD or catalase or both, were observed in all infertile subjects except those with normospermia (Fig. 2C and D). This is in agreement with the reported observation that free radicals of nitrogen are responsible for decreased activities of antioxidant enzymes in the semen of infertile men (31). Significantly high catalase activities in the leukocytospermia group observed in our study may be a compensatory mechanism producing more free radical scavengers to maintain Fertility and Sterility â 139

6 normal sperm motility. This is supported by the finding that catalaselike rather than SOD-like activity was significantly higher in reactive oxygen species producing semen samples (32). Examination of semen includes mainly sperm motility and concentration. Sperm function tests, considered specialized tests, are not routinely carried out. The present study has shown that sperm function (Fig. 1) in terms of hypo-osmotic swelling, acrosomal status, and nuclear chromatin decondensation may be affected independently of motility in certain groups of infertile subjects, such as those with oligospermia and leukocytospermia; the possible reason could be free radical stress coming from either oxygen or nitrogen. Accordingly, the justification of simultaneous assessment of sperm function and reactive oxygen species in different groups of infertile subjects was reported earlier (33). However, the present findings do not support a similar assessment of NO for detection of sperm pathologies because the degree and nature of correlation with sperm function varies widely with different groups of infertile subjects. When sperm function was correlated statistically with total NO, a significant negative correlation was observed in infertile subjects with subnormal sperm motility (asthenospermia and leukocytoasthenospermia). Identical findings were reported by Balercia et al. (13), who observed a significant linear negative correlation between NO concentration and percentage sperm motility. In the present study, we examined for the first time the correlation of NO to sperm parameters (functions) other than motility. In proven-fertile controls, the correlation was linear, positive, and significant (r ¼ 0.603, P<.001), whereas in normospermic infertile men the association was negative but not significant (r ¼ 0.126, P<.231). This implies that in the normospermia group, NO levels are slightly beyond the physiologic limit. In the oligospermia and oligoasthenospermia groups, the correlation (Fig. 4) between sperm function and NO levels was significantly positive (r ¼ 0.5 and 0.6, respectively), indicating that subnormal sperm function as observed in these groups might be due to insufficient levels of NO present in the semen. Because reduced arginine availability may lead to reduced NO synthesis (34), therapeutic treatments with exogenous arginine may be considered as an option in such cases. On the other hand, in asthenospermia and leukocytospermia, in which a negative association of NO with sperm function is observed, treatment with antioxidants may prove beneficial, which needs to be confirmed by future studies. Acknowledgment: The authors thank Mohammed Ibrahim, Senior Laboratory Assistant, for carrying out routine semenology and sperm function tests. REFERENCES 1. 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