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1.  Altered Antioxidant Status and Increased Lipid Per-Oxidation in Seminal Plasma of Tunisian Infertile Men 
Human seminal plasma is a natural reservoir of antioxidants that protect spermatozoa from oxidative damages. There is evidence in literature supports the fact that impairments in seminal antioxidant and lipid per-oxidation status play important roles in the physiopathology of male infertility. Our present study forms the first one which was carried out in Tunisia. We evaluated the antioxidant status in the seminal plasma of 120 infertile men programmed to In Vitro Fertilization (IVF) for the first tentative. Patients were characterized by an idiopathic infertility. They were divided into three groups: normozoospermics who were considered as controls (n=40), asthenozoospermics (Astheno; n=45) and oligoasthenoteratozoospermics (OAT; n=35). Seminal activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) and the levels of glutathione (GSH), zinc (Zn) and malondialdehyde (MDA) were measured. With the significant increase of the seminal activities of SOD and GPX in normozoospermics group, there were positive correlations observed between this enzymes and sperm quality. Also, significant elevated rates of seminal zinc and GSH were observed in control group, but there was contradictory associations reflecting the effects of these antioxidants on semen parameters. However, we noted significant increase of MDA levels in groups with abnormal seminogram. We showed negative associations between this per-oxidative marker and sperm parameters. These results obviously suggested that impairment on seminal antioxidants is an important risk factor for low sperm quality associated to idiopathic infertility and as a result can lead to poor IVF outcome.
PMCID: PMC3248656  PMID: 22211112
Oxidative damage; Antioxidant enzymes; Semen quality; Male infertility; Sperm abnormalities; lipid per-oxidation.
2.  Lipid Peroxidation and Nitric Oxide Levels in Male Smokers' Spermatozoa and their Relation with Sperm Motility 
Nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as nitric oxide synthases. Low concentrations of NO is essential in biology and physiology of spermatozoa, but high amounts of NO is toxic and has negative effects on sperm functions. Moreover, sperm membrane contains high concentrations of polyunsaturated fatty acids that are highly susceptible to oxidative damage that interferes with fertilization ability. Therefore, we investigated the correlation between levels of sperm malondialdehyde (MDA) and NO with sperm motility in male smokers.
Semen samples were collected from normozoospermic smoker (n=64) and nonsmoker (n=83) men. The content of sperm lipid peroxidation was determined by measuring malondialdehyde (MDA). The sperm NO were also measured using Griess reagent. Data was analyzed by SPSS, (version 15.0), using independent t-test and Pearson analysis.
The mean MDA and NO concentrations in the sperm of normozoospermic male smokers were significantly higher than the control group or normozoospermic nonsmokers, (p <0.001). A significant negative relationship was noted between sperm motility and sperm MDA levels (r=−0.32, p=0.01); and sperm motility and sperm NO concentration (for nitrite, r=−0.34, p=0.006 and for nitrate, r=−0.38, p=0.002).
It was concluded that the increase in MDA and NO production in sperm can influence sperm motility in normozoospermic smokers. Therefore, it seems that cigarette smoking may affect the fertility of male smokers via increasing the amount of sperm MDA/lipid peroxidation and NO concentrations.
PMCID: PMC3719338  PMID: 23926529
Cigarette smoking; Human sperm; Lipid peroxidation; Nitric oxide; Smoker men
3.  Correlation of Sperm Parameters With Semen Lipid Peroxidation and Total Antioxidants Levels in Astheno- and Oligoasheno- Teratospermic Men 
Sperm dysfunction caused by reactive oxygen species (ROSs) is one of the major causes of infertility in men, which leads to, lipid peroxidation (LPO) and the formation of stable peroxidation products like Malondialdehyde (MDA) in seminal plasma. MDA is effective factor in reducing fertility.
The aim of this study is to determine two biochemical markers of oxidative stress; TAC and MDA, and them correlation to quality-quantity factors in Asthenoteratospermic and Oligoashenoteratospermic men.
Patients and Methods
A total of 42 semen samples including: 15 samples normospermic as control group, 12 Asthenoteratospermic and 15 oligoasthenoteratospermic were collected from Babol IVF center; Iran. Semen analysis was performed according to WHO (1999) guidelines. Seminal plasma TAC and MDA levels in all patients were measured by TBARs and FRAP methods, respectively.
Seminal plasma TAC level in normospermic men was significantly higher than asthenoteratospermic men (P < 0.001) and oligasthenoteratospermic men (P < 0.001) and had posetive correlation with sperm count, motility and morphology. In contrast MDA levels in normospermic men were significantly lower than in asthenoteratospermic men (P = 0.049) and oligoasthenoteratospermic men (P = 0.001) and had negative correlation with sperm count, motility and morphology.
These results suggest that lipid peroxidation and decreasing total antioxidant capacity lead to low motility; morphology and sperm count in spermatozoa of astheno-and oligoastheno-teratospermic men. Therefore, evaluation of oxidative status and antioxidant defenses system may be as a useful tool for diagnosis and treatment of male infertility especially in idiopathic male infertility.
PMCID: PMC3929810  PMID: 24616785
Lipid Peroxidation; Malondialdehyde; Reactive Oxygen Species
4.  Total Antioxidant Capacity and Lipid Peroxidation in Semen of Patient with Hyperviscosity 
Cell Journal (Yakhteh)  2015;16(4):554-559.
Semen hyperviscosity (SHV) is one of the factors involved in deficiency in sperm function. This research aimed to evaluate seminal plasma total antioxidant capacity (TAC) and malondialdehyde (MDA) levels in infertile patients with hyperviscous and non-hyperviscous semen samples to understand whether hyperviscous semen is associated with oxidative damage in infertile subjects. In this cross sectional study, 59 semen samples were provided by fertile (n=12) individuals as control, infertile patients with normal viscosity (n=25) and infertile patients with hyperviscosity (n=22). After semen parameters examination, semen viscosity was studied by glass pipettes. Seminal plasma TAC and MDA levels were measured by ferric reducing of antioxidant power (FRAP) and thiobarbituric acid reaction (TBAR) methods, respectively. A probability less than 0.05 was considered statistically significant throughout the article. The mean of sperm parameters including: counts, motility and normal morphology in patients with hyperviscosity were significantly lower than those in non-hyperviscosity patients (p<0.05, p<0.01 and p<0.001, respectively). The mean of seminal plasma TAC value in seminal plasma of non-hyperviscosity patients (1710.31 ± 458.67 µmol/l) was significantly (p<0.01) higher than that of hyperviscosity group (1230.25 ± 352 µmol/l). A trend toward a higher mean of seminal plasma MDA value was estimated for hyperviscous group compared with non-hyperviscous (1.01 ± 0.41 nmol/ml vs. 0.94 ± 0.28 nmol/l); however, it was nonsignificant. Hyperviscous semen impairs seminal plasma TAC which is eventually associated with sperm membrane lipid peroxidation.
PMCID: PMC4297494
Male Infertility; Antioxidants; Lipid Peroxidation; MDA
5.  Seminal plasma levels of 15-F2α-isoprostane, malondialdehyde and total homocysteine in normozoospermic and asthenozoospermic males 
It has been proposed that oxidative stress plays an important role in male infertility. The aims of this study were to compare seminal plasma levels of 15-F2t-isoprostane (8-iso-PGF2α), malondialdehyde (MDA), and total (sum of free and bound) homocysteine (tHcy) from normozoospermic vs. asthenozoospermic men, and to examine the relationships between tHcy and lipid peroxidation products. The study was a case-control study with a simple random sampling. The case group was consisted of 15 asthenozoospermic males. This group was compared with 15 normozoospermic men. Seminal plasma levels of 15-F2α-isoprostane and tHcy were measured using commercially available enzyme immunoassay (EIA) kits. MDA levels were determined by the thiobarbituric acid (TBA) assay. The Mann-Whitney U test was used to compare two groups. Coefficients of correlation were calculated using Spearman’s correlation analysis. All hypothesis tests were two-tailed with statistical significance assessed at the p value <0.05 level. MDA levels were higher in asthenozoospermic subjects than in control subjects (0.72±0.06 μM vs. 0.40±0.06 μM; p<0.05). No differences were seen in 15-F2α-isoprostane levels in asthenozoospermic subjects and controls (65.00±3.20 pg/ml vs. 58.17±4.12 pg/ml; p>0.05). Interestingly, tHcy levels were to be slightly higher in asthenozoospermic subjects than in controls (6.18±1.17 μM vs. 4.8±0.52 μM). Sperm motility was inversely correlated with seminal plasma 15-F2α-isoprostane and MDA levels, respectively (p<0.05). In summary, seminal plasma levels of 15-F2α-isoprostane and tHcy showed no significant difference between normozoospermic and asthenozoospermic men. Sperm motility was not correlated with seminal plasma levels of tHcy. No relationship was found between tHcy and lipid peroxidation.
PMCID: PMC3453847  PMID: 23105539
15-F2α-isoprostane; Asthenozoospermia; Homocysteine; Lipid peroxidation; Malondialdehyde; Normozoospermia; Seminal plasma
6.  Role of ascorbic acid in human seminal plasma against the oxidative damage to the sperms 
This study evaluated the extent of oxidative stress by measuring malondialdehyde and ascorbic acid in the seminal plasma of human subjects with different fertility potential. Semen samples from 148 subjects were evaluated (48 normozoospermics, 34 oligoasthenoteratozoospermics, 34 asthenoteratozoospermics and 32 azoospermics). malondialdehyde level was found to be significantly higher in the abnormal groups (oligoasthenoterato and asthenoterato-zoospermics) than normozoospermics (P < 0.01). Negative correlation was also found between malondialdehyde level, sperm concentration, sperm motility and sperm morphology. Level of ascorbic acid was found to be significantly higher in normozoospermics than other abnormal groups (P < 0.01). It was found to be correlated positively with all seminogram parameters and negatively with malondialdehyde level. The study revealed that, excess lipid peroxidation reflected by high malondialdehyde level with reduced ascorbic acid in human seminal plasma is associated with poor semen quality where as ascorbic acid content has positive correlation with fertility potential.
PMCID: PMC3453303  PMID: 23105855
Ascorbic acid; Infertility; Oxidative stress; Human Seminal Plasma
7.  Relationship between seminal plasma zinc concentration and spermatozoa–zona pellucida binding and the ZP-induced acrosome reaction in subfertile men 
Asian Journal of Andrology  2009;11(4):499-507.
The aim of this study was to determine the relationship between seminal zinc concentration and spermatozoa–zona pellucida (ZP) binding and the ZP-induced acrosome reaction (ZPIAR) in subfertile men. Semen analyses and seminal zinc concentration assessments were carried out according to the World Health Organization manual for 458 subfertile men. A spermatozoa–ZP interaction test was carried out by incubating 2 × 106 motile spermatozoa with a group of four unfertilized oocytes obtained from a clinical in vitro fertilization programme. After 2 h of incubation, the number of spermatozoa bound per ZP and the ZPIAR of ZP-bound spermatozoa were examined. The effect of adding 0.5 mmol L−1 zinc to the media on the ZPIAR of spermatozoa from normozoospermic men was also tested in vitro. Seminal zinc concentration positively correlated with sperm count and duration of abstinence, but negatively correlated with semen volume. On analysis of data from all participants, both spermatozoa–ZP binding and the ZPIAR were significantly correlated with sperm motility and normal morphology, but not with seminal zinc concentration. However, in men with normozoospermic semen, the seminal zinc concentration was significantly higher in men with defective ZPIAR (< 16%) than in those with normal ZPIAR (≥ 16%) (P < 0.01). The addition of 0.5 mmol L−1 zinc to the culture media had no effect on spermatozoa–ZP binding, but significantly reduced the ZPIAR in vitro (P < 0.001). In conclusion, seminal zinc concentration is correlated with sperm count and the duration of abstinence in subfertile men. In men with normozoospermic semen, high seminal zinc concentration may have an adverse effect on the ZPIAR.
PMCID: PMC3735308  PMID: 19434054
semen analysis; seminal zinc; spermatozoa–zona pellucida interaction; subfertile men
8.  Assessment of the level of trace element zinc in seminal plasma of males and evaluation of its role in male infertility 
Male infertility is a serious problem all over the world. Nutritional deficiency of trace element Zinc (Zn) may play a role in male infertility as Zn plays an important role not only in normal testicular development, but also in spermatogenesis and sperm motility. Deficiency of Zn is associated with hypogonadism and insufficient development of secondary sex characteristics.
The present study was designed to analyze the level of seminal Zn among different groups of infertile patients and to correlate it with sperm concentration, active, sluggish and immotile fractions of seminal parameters, with an objective to establish the role of Zn in male infertility.
Setting and Design:
The present study was carried out in five- years period from 2004 to 2009. It was a descriptive analytical study with non probability sampling.
Materials and Methods:
Semen examination of the patients was carried out according to the standardized method of the World Health Organization. Semen Zn was estimated by color 5 Br. PAPS method.
Statistical Analysis:
All statistical analyses were performed by using SPSS (Version 14.0 for windows) software, by applying student's t-test.
The result showed that seminal Zn was 702.92±10.60, 598.48±12.95, 617.54±9.55, 542.29±22.75, 710.36±7.87, 712.06±7.96, 789.36±21.33, and 762.06±8.99 mg/dl in azoospermic, oligozoospermic, asthenozoospermic, oligoasthenozoospermic, teratozoospermic, normozoospermic, polyzoospermic, and proven fathers group, respectively.
Decreased concentration of seminal Zn do affect the sperm count, while increased level of seminal plasma Zn causes decreased sperm motility; so, it is suggested that administration of Zn should be very carefully monitored in such patients having low sperm count but normal sperm motility, as adequate seminal Zn is required for normal sperm function.
PMCID: PMC3657968  PMID: 23776785
Asthenozoospsermia; azoospermia; male infertility; oligozoospermia; seminal zinc
9.  Sperm DNA fragmentation and oxidation are independent of malondialdheyde 
There is clinical evidence to show that sperm DNA damage could be a marker of sperm quality and extensive data exist on the relationship between DNA damage and male fertility status. Detecting such damage in sperm could provide new elements besides semen parameters in diagnosing male infertility. We aimed to assess sperm DNA fragmentation and oxidation and to study the association between these two markers, routine semen parameters and malondialdehyde formation.
Semen samples from 55 men attending the Histology-Embryology Laboratory of Sfax Faculty of Medicine, Tunisia, for semen investigations were analysed for sperm DNA fragmentation and oxidation using flow cytometry. The Sperm was also assessed spectrophotometrically for malondialdehyde formation.
Within the studied group, 21 patients were nonasthenozoospermic (sperm motility ≥ 50%) and 34 patients were considered asthenozoospermic (sperm motility < 50%). A positive correlation was found between sperm DNA fragmentation and oxidation (p = 0.01; r = 0.33). We also found a negative correlation between sperm DNA fragmentation and some sperm parameters: total motility (p = 0.001; r = -0.43), rapid progressive motility (type a motility) (p = 0.04; r = -0.27), slow progressive motility (type b motility) (p = 0.03; r = -0.28), and vitality (p < 0.001; r = -0.65). Sperm DNA fragmentation was positively correlated with coiled tail (p = 0.01; r = 0.34). The two parameters that were found to be correlated with oxidative DNA damage were leucocytes concentrations (p = 0.01; r = 0.38) and broken neck (p = 0.02; r = 0.29). Sperm MDA levels were negatively correlated with sperm concentration (p < 0.001; r = -0.57), total motility (p = 0.01; r = -0.35) and type a motility (p = 0.03; r = -0.32); but not correlated with DNA fragmentation and DNA oxidation.
Our results support the evidence that oxidative stress plays a key role in inducing DNA damage; but nuclear alterations and malondialdehyde don't seem to be synchronous.
PMCID: PMC3098153  PMID: 21492479
10.  Elemental composition of human semen is associated with motility and genomic sperm defects among older men 
Older men tend to have poorer semen quality and are generally at higher risks for infertility and abnormal reproductive outcomes.
We employed proton-induced X-ray emission (PIXE, 3 MeV proton beam) to investigate the concentrations of zinc, copper, calcium, sulfur, chlorine, potassium, titanium, iron and nickel in washed sperm and seminal plasma from non-smoking groups of 10 older men (65–80 years old) and 10 younger men (22–28 years old) who were concurrently assayed for sperm function and genomicly defective sperm.
The older group showed elevated zinc, copper and calcium in sperm and elevated sulfur in seminal plasma compared with the younger men. The older group also showed reduced motility as well as increased sperm DNA fragmentation, achondroplasia mutations, DNA strand breaks and chromosomal aberrations. Sperm calcium and copper were positively associated with sperm DNA fragmentation (P < 0.03). Seminal sulfur was positively associated with sperm DNA fragmentation and chromosomal aberrations (P < 0.04), and negatively associated with sperm motility (P < 0.05). Sperm calcium was negatively associated with sperm motility, independent of male age (P = 0.01).
We identified major differences in elemental concentrations between sperm and seminal plasma and that higher sperm copper, sulfur and calcium are quantitatively associated with poorer semen quality and increased frequencies of genomic sperm defects.
PMCID: PMC3619967  PMID: 23042799
male aging; calcium; copper; semen; zinc
11.  Effect of antioxidants and antibiotics on levels of seminal oxidative stress in leukocytospermic infertile men 
Defective sperm function is the most common cause of infertility. A prospective study was carried out to correlate the concentration of nitrite (the stable metabolite of nitric oxide) in seminal plasma with leukocytospermia, and sperm membrane integrity. Total Fifty-seven normozoospermic subjects with and without leukocytospermia visiting the Infertility clinic at KH and MRC, Karad, were included in the present study. Semen samples were checked for sperm concentration, total sperm count, sperm motility, seminal leukocyte concentration and sperm membrane integrity as Hypoosmotic Swelling Test. Similarly the concentration of nitrite in seminal plasma was measured by Griess reaction and total antioxidant power measured as ferric reducing ability of plasma. The concentration of nitrite in seminal plasma was found to be raised with significantly increased leukocyte concentration in semen. Also significantly lowered levels of total antioxidant power along with defective sperm function was observed. Our results suggest that supplementary treatment of antioxidants with antibiotic for leukocytospermic infertile male patients may improve the sperm membrane integrity.
PMCID: PMC3453748  PMID: 23105588
Leukocytes and Total antioxidant power; Nitric oxide (as a nitrite); Sperm membrane integrity
12.  Sperm motility inversely correlates with seminal leptin levels in idiopathic asthenozoospermia 
Background: Asthenozoospermia is one kind cause of male infertility. Nevertheless, no specific etiology can be identified by routine tests in some cases. Recently, it has been shown that leptin plays a critical role in male fertility. However, the link between leptin and sperm motility is yet to be determined. The aim of this study was to explore association between seminal and serum leptin levels and sperm motility in idiopathic asthenozoospermia. Methods: Our study included 79 asthenozoospermic men and 77 normozoospermic men. Semen was assessed by volume, sperm concentration, motility and morphology. Serum gonadotropic and sex hormones were determined by a chemiluminescent assay. The leptin levels in serum and seminal plasma were detected with ELISA. Results: The mean seminal leptin level in asthenozoospermic group was significantly higher than that in control group, but there was no significant difference in the serum leptin levels between these two groups. The serum leptin had no significant correlation with sperm motility. The seminal leptin had significantly negative correlation with sperm progressive motility and serum total testosterone. Conclusions: The findings indicate a pathophysiological relevance of seminal leptin in sperm motility.
PMCID: PMC4238556  PMID: 25419396
Idiopathic asthenozoospermia; seminal leptin; serum leptin; sperm motility
13.  Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility 
Objectives: Reactive oxygen species (ROS) induced lipid peroxidation is associated with sperm function. Malondialdehyde (MDA) concentration and glutathione peroxidase (GPx) activity represent the lipid peroxidation and spermicidal antioxidant, respectively. We aimed to evaluate the relationship of MDA and GPx levels with sperm parameters.
Patients and methods: Specimens were divided into two groups: group 1. normospermia (n=20); group 2. oligoasthenospermia (n=31). Seminal MDA concentration was measured by thiobarbituric acid reaction method. Seminal GPx activity was measured by oxidation of reduced nicotinamide-adenine dinucleotide. Seminal MDA levels and GPx activities in both groups were compared.
Results: MDA concentrations in both groups were significantly different (1.52 ± 0.75 vs. 2.25 ± 0.88 nM, p = 0.0021). GPx activities in both groups were non-significantly different (0.48 ± 0.11 vs. 0.47 ± 0.12 U/ml). MDA levels were negatively correlated with the sperm motility (MDA = -0.014 x motility + 2.62, p =0.017) and concentration (MDA = -0.0045 x concentration + 2.23, p = 0.0166). GPx activities were positively but non-significantly correlated with the sperm concentration and sperm motility.
Conclusions: Seminal MDA concentrations are negatively correlated with sperm concentration and motility, which might provide a simple and useful tool in predicting sperm parameters. GPx activity is non-significantly correlated with the seminal quality. Roles of seminal MDA upon spermatogenesis merits further surveys.
PMCID: PMC1457038  PMID: 16680200
glutathione peroxidase; malondialdehyde; oligoasthenozoospermia; semen; sperm
14.  The Association of Seminal Plasma Antioxidant Levels and Sperm Chromatin Status with Genetic Variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) in Infertile Men with Oligoasthenoteratozoospermia 
Disease markers  2013;34(3):205-210.
In this study we aimed to examine the effects of genetic variants of GSTM1 and GSTP1 (Ile105Val and Ala114Val) on GST activity, seminal oxidative stress and sperm chromatin status in infertile men with oligoasthenoteratozoospermia (OAT). The study population (n = 121) consisted of 95 infertile men with OAT and 26 controls with normozoospermia. Multiplex polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were utilized to detect the aforesaid genetic variants. We measured GST activity and total antioxidant capacity (TAC) of seminal plasma by spectrophotometry. Sperm chromatin integrity and maturity were assessed using toluidine blue and chromomycin A3 (CMA3-positive sperm) staining, respectively. The analysis showed that subgroups of GSTM1 null and GSTP1 C/T+T/T genotypes in comparison with GSTM1 present and GSTP1 wild type (C/C) genotypes did not have statistically significant differences in both OAT or normozoospermic men considering sperm concentration and motility, percentage of CMA3-positive sperm, seminal plasma TAC, sperm chromatin integrity and GST activity. Thus, the findings of our study suggest that there are no significant associations between GSTM1 and GSTP1 polymorphisms and sperm parameters at conventional or at molecular levels including OS status, sperm chromatin integrity or maturity in Iranian infertile men with OAT and normozoospermia. However, these polymorphisms could be related to the fertility status of the studied population but not evaluated in this study.
PMCID: PMC3809979  PMID: 23324581
GSTM1; GSTP1; normozoospermia; polymorphism; oligoasthenoteratozoospermia; sperm
15.  Seminal plasma levels of free 8-isoprostane and its relationship with sperm quality parameters 
There is growing evidence that damage to spermatozoa by reactive oxygen species play a key role in male infertility. The aim of this study was to assess seminal plasma free 8-Isoprostane levels in men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia compared to normozoospermic males and its correlation with seminal parameters. The case group consisted of men with asthenozoospermia (n=15), asthenoteratozoospermia (n=16) and oligoasthenoteratozoospermia (n=15). The control group consisted of 16 males with normozoospermia. After Purification of Free 8-Isoprostane by affinity column, its concentration was measured by enzyme immunoassay method. Free 8-Isoprostane evaluation showed significantly greater values in the total case group (n=46) versus control group (18.23±3.56 vs 2.6±0.38 ng/ml). In each case group free 8-Isoprostane also showed a significant increasing compared to normozoospermic males. Free 8-Isoprostane showed an inversely significant correlation with sperm motility and sperm morphology. Lipid peroxidation could have significant role in etiology of sperm abnormalities. Measurement of 8-Isoprostane can be used as a specific biomarker for assessing lipid peoxidation in sperm.
PMCID: PMC3453664  PMID: 23105720
Isoprostane; Reactive Oxygen Species; Lipid peroxidation; Semen; Sperm
16.  Influence of oxidative stress on functional integrity of human spermatozoal membrane 
To study the relationship between lipid peroxidation of spermatozoa and changes in functional intergrity of human spermatozoa membrane in male subjects.
Materials and Methods
Sixty eight male partners of infertile couples were included in the study. Status of oxidative stress was assessed by determining malondialdehyde (MDA) in seminal plasma. Functinal intergrity of sperm membrance was tested subjecting the sperm to hypo-osmotic test (HOS). The seminal plasma MDA levels were compared with seminogram parameters as well as with the results of HOS test using Pearson’s coefficient of correlation (r) and significance of coefficient of correlation calculated from the table.
A significant but weak negative correlation was observed between seminal plasma MDA level and sperm concentration (r=−0.33,p<0.05), sperm motility (r=−0.37,p<0.05), sperm morphology (r=−0.37,p<0.05), and percentage of HOS positive spermatozoa (r=−0.33,p<0.05). Percentages of HOS positive spermatozoa also exhibited a significant but weak negative relationship with sperm concentration (r=−0.47,p<0.01), sperm motility (r=−0.48,p<0.01), sperm morphology (r=−0.49,p<0.01).
Lipid peroxidation of spermatozoa is likely to affect the functional intergrity of the spermatozoa membrane.
PMCID: PMC3394549
oxidative stress; spermatozoa; membrane integrity
17.  Levels of oxidative stress biomarkers in seminal plasma and their relationship with seminal parameters 
There is growing evidence that damage to spermatozoa by reactive oxygen species (ROS) play a key role in male infertility. The aim of the present study was to assess seminal plasma levels of total antioxidant capacity (TAC), free 8-Isoprostane and activities of catalase and superoxide dismutase (SOD) in men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia compared with normozoospermic males.
The patients consisted of 46 men with seminal parameters abnormalities. The patients were grouped into asthenozoospermic (n = 15), asthenoteratozoospermic (n = 16) and oligoasthenoteratozoospermic (n = 15). The control group consisted of 16 healthy males with normozoospermia. Catalase activity was measured by Aebi spectrophotometeric method. Levels of TAC and SOD were measured by commercially available colorimetric assays. Level of free 8-Isoprostane was assessed by commercially available enzyme immunoassay (EIA) method. Differences between groups were assessed using Mann-Whitney U test and Kruskal-Wallis test. Coefficients of correlation were calculated using Spearman's correlation analysis. All hypothesis tests were two-tailed with statistical significance assessed at the p value < 0.05 level with 95% confidence intervals
Levels of catalase and TAC were significantly lower in patients than the control group. No significant changes were seen in SOD activities. Levels of free 8-Isoprostane were significantly higher in patients than the control group. Furthermore, asthenozoospermic, asthenoteratozoospermic and oligoasthenoteratozoospermic groups had significantly lower values of catalase activity and TAC when compared to normozoospermic males. Levels of free 8-Isoprostane were significantly higher in all patients subgroups than the control group. Levels of catalase and TAC were positively correlated with sperm motility and morphology. Free 8-Isoprostane levels showed an inverse correlation with sperm motility and morphology.
Decreasing seminal plasma antioxidants levels, especially catalase and TAC, could have significant role in etiology of impaired sperm function. Measurement of 8-Isoprostane may be used as a specific biomarker for assessing oxidative stress on sperm.
PMCID: PMC1906821  PMID: 17540046
18.  Mucuna pruriens Reduces Stress and Improves the Quality of Semen in Infertile Men 
The present investigation was undertaken to assess the role of Mucuna pruriens in infertile men who were under psychological stress. Study included 60 subjects who were undergoing infertility screening and were found to be suffering from psychological stress, assessed on the basis of a questionnaire and elevated serum cortisol levels. Age-matched 60 healthy men having normal semen parameters and who had previously initiated at least one pregnancy were included as controls. Infertile subjects were administered with M. pruriens seed powder (5 g day−1) orally. For carrying out morphological and biochemical analysis, semen samples were collected twice, first before starting treatment and second after 3 months of treatment. The results demonstrated decreased sperm count and motility in subjects who were under psychological stress. Moreover, serum cortisol and seminal plasma lipid peroxide levels were also found elevated along with decreased seminal plasma glutathione (GSH) and ascorbic acid contents and reduced superoxide dismutase (SOD) and catalase activity. Treatment with M. pruriens significantly ameliorated psychological stress and seminal plasma lipid peroxide levels along with improved sperm count and motility. Treatment also restored the levels of SOD, catalase, GSH and ascorbic acid in seminal plasma of infertile men. On the basis of results of the present study, it may be concluded that M. pruriens not only reactivates the anti-oxidant defense system of infertile men but it also helps in the management of stress and improves semen quality.
PMCID: PMC2816389  PMID: 18955292
antioxidants; lipid peroxides; male infertility; Mucuna pruriens; psychological stress
19.  The Total Antioxidant Power of Semen and Its Correlation with the Fertility Potential of Human Male Subjects 
Background: There are growing evidences that the damage which is caused to the spermatozoa by the Reactive Oxygen Species (ROS) plays a key role in the male infertility. The seminal plasma is endowed with many enzymatic and nonenzymatic antioxidants which protect the spermatozoa against oxidative stress.The present study was undertaken by using a simple, colourimetric, ferric reducing, antioxidant power for assessing the total antioxidant power rather than the individual antioxidants. The measurement of the individual antioxidants in the seminal plasma, such as Superoxide Dismutase, Vitamin E, etc. is time consuming, which often requires sophisticated and expensive techniques and these measurements may not correlate with the quality of semen.
Aim: To evaluate the total antioxidant capacity of seminal plasma by estimating the Ferric Reducing Antioxidant Power (FRAP) of semen in different groups of subjects and to correlate it with the different seminogram parameters.
Material and Methods: The semen samples were obtained from 150 male partners of infertile couples who attended the Reproductive Biology Unit (Infertility Clinic) of the Department of Physiology, MGIMS, Sevagram, who were aged 20-58 years and they were analyzed for the routine seminogram parameters. All the subjects were categorized into two main groups, A. The subjects with abnormal ejaculates, who were further sub classified into the following groups i) Asthenoteratozoospermics (n=25) ii) Oligoasthenoteratozoospermics (n=26) and iii) Azoospermics (n=19) and B. The subjects with normal ejaculates (n=80). The total antioxidant power was measured spectrophotometrically by using the Ferric Reducing Antioxidant Power (FRAP) assay.
Results: The Total Antioxidant Capacity (TAC) was found to be significantly lower in the abnormal ejaculates than in the normal ejaculates. A statistically significant positive correlation was observed between the TAC and all the seminogram parameters such as the sperm concentration, sperm motility and sperm morphology (p<0.05).
Conclusion: A decreased seminal plasma antioxidant capacity (TAC) could have significant role in the aetiology of impaired sperm functions. So, the tac may be used as specific biomarker for assessing the oxidative stress in sperms.
PMCID: PMC3708257  PMID: 23905087
Male infertility; Seminal plasma; Total Antioxidant Capacity (TAC); Ferric Reducing Antioxidant Power (FRAP)
20.  Study of the effects of oral zinc supplementation on peroxynitrite levels, arginase activity and NO synthase activity in seminal plasma of Iraqi asthenospermic patients 
Low concentrations of nitric oxide (NO) are necessary for the biology and physiology of spermatozoa, but high levels of NO are toxic and have negative effects on sperm functions. Although several studies have considered the relationship between infertility and semen NO concentrations, no study on the effects of asthenospermia treatments such as oral zinc supplementation on concentrations of NO, which are important in fertility, has been reported. Studies have shown that oral zinc supplementation develops sperm count, motility and the physical characteristics of sperm in animals and in some groups of infertile men. The present study was conducted to study the effect of zinc supplementation on the quantitative and qualitative characteristics of semen, along with enzymes of the NO pathway in the seminal plasma of asthenospermic patients.
Semen samples were obtained from 60 fertile and 60 asthenozoospermic infertile men of matched age. The subfertile group was treated with zinc sulfate; each participant took two capsules (220 mg per capsule) per day for 3 months. Semen samples were obtained (before and after zinc sulfate supplementation). After liquefaction of the seminal fluid at room temperature, routine semen analyses were performed. The stable metabolites of NO (nitrite) in seminal plasma were measured by nitrophenol assay. Arginase activity and NO synthase activity were measured spectrophotometrically.
Peroxynitrite levels, arginase activity, NO synthase activity and various sperm parameters were compared among fertile controls and infertile patients (before and after treatment with zinc sulfate). Peroxynitrite levels and NO synthase activity were significantly higher in the infertile patients compared to the fertile group. Conversely, arginase activity was significantly higher in the fertile group than the infertile patients. Peroxynitrite levels, arginase activity and NO synthase activity of the infertile patient were restored to normal values after treatment with zinc sulfate. Volume of semen, progressive sperm motility percentage and total normal sperm count were increased after zinc supplementation.
Treatment of asthenospermic patients with zinc supplementation leads to restored peroxynitrite levels, arginase activity and NO synthase activity to normal values and gives a statistically significant improvement of semen parameters compared with controls.
Trial registration identifier: NCT01684059
PMCID: PMC3882288  PMID: 24383664
Asthenospermia; Nitric oxide synthase; Arginase; Peroxynitrite; Zinc; Zinc supplementation; Oxidative stress
21.  Ascorbic Acid in Human Seminal Plasma: Determination and Its Relationship to Sperm Quality 
The objective of the present study was to assess the ascorbic acid (AA) levels in seminal plasma of the fertile and infertile men and to investigate its relationship with sperm count, motility and normal morphology. Semen samples were provided by fertile [smoker (n = 25), nonsmoker (n = 21)] and infertile men [smoker (n = 23), nonsmoker (n = 32)]. A simplified method of reverse phase high performance liquid chromatography (RP-HPLC) procedure using UV detection was applied for the determination of seminal AA. Fertile subjects, smoker or not, demonstrated significantly higher seminal AA levels than any infertile group (p<0.01). Nonsmokers had high, but no significant, mean AA levels in their seminal plasma compared with smokers. Seminal AA in fertile and infertile (smokers or nonsmokers) males correlated significantly with the percentage of spermatozoa with normal morphology (p<0.01). Seminal AA decreased significantly in infertile men. Decrease of seminal plasma AA is a risk factor for low normal morphology of spermatozoa and idiopathic male infertility. Measurement of seminal AA in the seminal plasma of males with a history of subfertility or idiopathic infertility is necessary and can be helpful in fertility assessment.
PMCID: PMC2735625  PMID: 19794921
ascorbic acid; sperm quality; seminal plasma; male infertility; RP-HPLC
22.  Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men 
PLoS ONE  2013;8(7):e68490.
Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods.
The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/106 dG were respectively 7.85 and 5.87 (p = 0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.
PMCID: PMC3709910  PMID: 23874641
23.  Evaluation of Serum Testosterone, Progesterone, Seminal Antisperm Antibody, and Fructose Levels among Jordanian Males with a History of Infertility 
Due to the biochemical complexity of seminal fluid, we attempt to study the possible correlation between fructose, which is secreted under the effect of androgen hormone, and autoimmunity, which might play a role in varicocele associated infertility, in reducing sperm motility. Seminal fructose, antisperm antibodies (ASAs) and blood steroids hormones (testosterone and progesterone) levels were measured in 66 infertile males with varicocele and 84 without varicocele referred for fertility treatment. Seminal analysis was performed with biochemical measurements of seminal fructose and mixed agglutination reaction (MAR) for ASA. Serum levels of progesterone and testosterone were estimated using a competitive chemoluminescent enzyme immunoassay. The mean values for serum testosterone were 380.74 ± 24.331, 365.9 ± 16.55, and 367.5 ± 21.8 ng/dl, progesterone 0.325 ± 0.243, 0.341 ± 0.022, and 0.357 ± 0.0306 ng/ml, and seminal plasma fructose 359.6 ± 26.75, 315.6 ± 13.08, and 332.08 ± 24.38 mg/dl in males with varicocele, without varicocele, and fertile males, respectively. A significant high level of testosterone was observed within varicocele group (P = .001). This result showed that testosterone may play a role as an infertility determinant in subjects with varicocele. ASA was detected in 18 (26.47%) of cases with varicocele, 20 (38.46%) without varicocele, and in 16 (32.0%) fertile men. Cases with ASAs associated with low sperm motility morphology. An inverse correlation between sperm-bound antibodies and viscosity has been shown (P = .017). ASA showed some significant inverse relations with ages, durations of infertility, and viscosity (P < .05). In addition, a significant correlation was observed between ASA positive seminal plasma and testosterone concentration among infertile cases (with or without varicocele) and fertile (P < .05). Our results suggest a relationship between testicular steroid hormone levels with autoimmunity and sperm antibodies which influence the motility of ejaculated spermatozoa among Jordanian infertile males.
PMCID: PMC3003948  PMID: 21188073
24.  Evaluation of CD52 positive sperms in subfertile human semen samples: Is there any relationship with main semen parameters? 
Sperm maturation and sperm membrane integration are the most important elements in male fertility. CD52 is one of the antigens. CD52 is a GPI (glycosylphosphatidylinositol) anchored that express on lymphocytes and epididymal cells. This antigen bind to sperm membrane during transition sperm from epididymal duct as well as its relationship with semenogelins in human seminal plasma. The aim of this study was to obtain any association between the percentage of CD52 positive sperms with main semen parameters such as percentage of motile sperms, percentage of sperm with normal morphology, and the presence of normal viscosity.
Materials and Methods:
Semen samples from subfertile men were analyzed, the samples totally were 45 that divided according to their motility into three groups, first one, more than 40%, second one 10-40%, and the third one under 10% total motility. Fifteen samples in each group were evaluated by semen analysis according to WHO 2010 guidelines for infertility laboratory. Sperms were washed by Ham's F-10 and immunostaining with the monoclonal antibody CAMPATH-1G and then analyzed by flow cytometry. We compared each of the groups based on their motility and the data were analyzed by SPSS 20.
Correlation between CD52 labeling and sperm motility was negatively significant, in the second group (r = –0.592, P = 0.020) and in the third group (r = –0.805, P = 0.00).
Our results showed that the correlation between CD52 labeling and sperm motility was negatively significant, but we did not observe any relation with other semen parameters, such as sperm normal morphology, sperm concentration, and semen viscosity.
PMCID: PMC4063091  PMID: 24949295
CD52; infertility; sperm parameters
25.  Evaluation of atorvastatin efficacy and toxicity on spermatozoa, accessory glands and gonadal hormones of healthy men: a pilot prospective clinical trial 
Recommendations for cardiovascular disease prevention advocate lowering both cholesterol and low-density lipoprotein cholesterol systemic levels, notably by statin intake. However, statins are the subject of questions concerning their impact on male fertility. This study aimed to evaluate, by a prospective pilot assay, the efficacy and the toxicity of a decrease of cholesterol blood levels, induced by atorvastatin on semen quality and sexual hormone levels of healthy, normocholesterolaemic and normozoospermic men.
Atorvastatin (10 mg daily) was administrated orally during 5 months to 17 men with normal plasma lipid and standard semen parameters. Spermatozoa parameters, accessory gland markers, semen lipid levels and blood levels of gonadal hormones were assayed before statin intake, during the treatment, and 3 months after its withdrawal.
Atorvastatin treatment significantly decreased circulating low-density lipoprotein cholesterol (LDL-C) and total cholesterol concentrations by 42% and 24% (p < 0.0001) respectively, and reached the efficacy objective of the protocol. During atorvastatin therapy and/or 3 months after its withdrawal numerous semen parameters were significantly modified, such as total number of spermatozoa (-31%, p < 0.05), vitality (-9.5%, p < 0.05), total motility (+7.5%, p < 0.05), morphology (head, neck and midpiece abnormalities, p < 0.05), and the kinetics of acrosome reaction (p < 0.05). Seminal concentrations of acid phosphatases (p < 0.01), α-glucosidase (p < 0.05) and L-carnitine (p < 0.05) were also decreased during the therapy, indicating an alteration of prostatic and epididymal functions. Moreover, we measured at least one altered semen parameter in 35% of the subjects during atorvastatin treatment, and in 65% of the subjects after withdrawal, which led us to consider that atorvastatin is unsafe in the context of our study.
Our results show for the first time that atorvastatin significantly affects the sperm parameters and the seminal fluid composition of healthy men.
Trial registration NCT02094313.
PMCID: PMC4114109  PMID: 25016482
Atorvastatin; Human Spermatozoa; Seminal fluid; Cholesterol; Gonadotropins; Testosterone; Normocholesterolemic; Normozoospermic; Healthy men

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