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1.  Toll-like receptors (TLR) 2 and 4 on human sperm recognize bacterial endotoxins and mediate apoptosis 
Human Reproduction (Oxford, England)  2011;26(10):2799-2806.
Bacterial infections of the genital tract are one of the most serious causes of infertility in males. In some human patients with poor semen quality, leukocytospermia has been observed. Because leukocytes express the bacterial-lipopolysaccharide (LPS) responsive Toll-like receptor (TLR) signaling cascade and secrete tumor necrosis factor-α, secreted cytokines comprise one, but probably not the only, class of factors that can impact sperm motility.
In this study, we documented that bacterial endotoxins, LPS and peptidoglycan, can be detected in human semen. Furthermore, the addition of endotoxins in the absence of leukocytes directly and significantly reduced the motility and increased the apoptotic rate of both human and mouse sperm and suppressed fertilization by mouse sperm both in vivo and in vitro. The well-known LPS receptor, TLR4, and peptidoglycan receptor, TLR2, were expressed in human and mouse sperm. In Tlr2/4−/− double-mutant mice, the negative effects of endotoxins on sperm functions were blocked, suggesting that the bacterial endotoxins mediated activation of TLR-dependent pathways in sperm leading to apoptosis.
Sperm can recognize bacterial endotoxins by TLRs present in their membranes. The activated TLRs reduce sperm motility, induce sperm apoptosis and significantly impair the potential for fertilization.
PMCID: PMC3174031  PMID: 21775336
peptidoglycan; lipopolysaccharide; sperm motility; sperm apoptosis; Toll-like receptors
2.  Effect of sesame on sperm quality of infertile men 
High level of semen reactive oxygen species is considered as an important factor in male infertility. Sesame has antioxidant properties, which could be effective on improvement of semen parameters. This study was designed to determine the effects of sesame on sperm quality.
Materials and Methods:
Twenty-five infertile men entered this clinical trial. They were treated with a 3-months course of taking 0.5 mg/kg sesame. The pre intervention sperm analysis (sperm count, motile sperm percentage and normal morphology sperm percentage) was compared with post treatment sperm analysis. Based on the post intervention seamen analysis, patients were advised to undergo either IVF or ICSI to assess their fertility status.
There was significant improvement in the sperm count (10.56 ± 5.25 vs. 22.71 ± 30.14 million per ml) and motility (15.32 ± 13.58 vs. 23.32 ± 20.61 percent) after treatment with sesame (P value: 0.04 and <0.0001 respectively), but there was no significant improvement in sperm morphology after the treatment (10.72 ± 6.66 vs. 13.20 ± 11.14 percent, P value: 0.10). Three patients (12%) underwent IUI, which resulted in 1 successful pregnancy. Two patients (8%) underwent ICSI, which was not successful; however 2 (8%) patients had spontaneous pregnancy. Fortunately, all pregnancies led to live birth. Except 1 case of diarrhea, no other major side effect was reported.
Sesame improved sperm count and motility, and can be prescribed as an effective and safe method for male factor infertility.
PMCID: PMC3732896  PMID: 23930112
Male infertility; infertility; semen quality; sesame
3.  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
4.  Seminal bacterial contaminations: Probable factor in unexplained recurrent pregnancy loss  
Background: It is estimated that about 50% of causes of recurrent pregnancy loss (RPL) cases remain unknown. Sperm factors are suggested to have probable role in cases with RPL.
Objective: The goal was to determine the possible relationship between semen bacterial contaminations with unexplained RPL. Also, the correlation between number of bacterial colony and sperm chromatin condensation was examined.
Materials and Methods: This study consisted of 30 fertile men (group A) and 30 infertile (group B) men with unknown RPL. Semen collection and analysis were done according to WHO manuals. Sperm count and motility were evaluated by Makler chamber. Eosin-Nigrosin and Papanicolaou staining methods were applied for viability and morphology assessment, respectively. The semen samples from both groups were cultured for aerobic bacteria. Aniline blue (AB) and toluidine blue (TB) staining methods were applied for evaluating sperm chromatin condensation.
Results: The numbers of colonies were significantly higher in group B when compared to group A. Also, S. aureus and E. coli contaminations showed significant differences between two groups. Both AB+ and TB+ sperm cells showed significant increase in group B compared to group A. There was a significant negative correlation between colony number and progressive motility (p=0.01), and sperm viability (p=0.007). In addition, positive correlations were found between colony number and AB+ (p=0.001) and TB+ (p=0.004) as well.
Conclusion: Bacterial contaminations in semen of men from RPL couples had significantly higher levels when compared to fertile controls. Presence of microorganisms in semen may be correlated with irregular sperm parameters and quality.
PMCID: PMC3941390  PMID: 24639718
Recurrent pregnancy loss; Bacteria; Semen
5.  Eurycoma longifolia Jack in managing idiopathic male infertility 
Asian Journal of Andrology  2010;12(3):376-380.
This study investigated the effect of treatment with the proprietary standardized, water-soluble extract of the root of the Malaysian plant, Eurycoma longifolia Jack, which is thought to enhance male fertility with regard to higher semen volumes, sperm concentrations, the percentage of normal sperm morphology and sperm motility in male partners of sub-fertile couples with idiopathic infertility. A total of 350 patients were given 200 mg of the extract daily and follow-up semen analyses were performed every 3 months for 9 months. Of these 350 patients, 75 patients completed one full cycle of 3 months. Follow-up semen analyses in these patients showed significant improvement in all semen parameters. The proprietary extract of Eurycoma longifolia Jack significantly improved the sperm quality in these patients, allowing for 11 (14.7%) spontaneous pregnancies.
PMCID: PMC3739276  PMID: 20348942
male infertility; medicinal herbs; semen
6.  Semen quality in men from subfertile couples from the region of Łódź (Poland) according to the new reference values recommended by WHO 2010 
The semen analysis is the main diagnostic tool for evaluating the male fertility potential. The standard semen analysis includes evaluation of the sperm concentration, motility, and their morphology. The most important question is whether the results from semen analysis may be accurate markers for male fertility. Therefore, we retrospectively studied sperm quality among men attending the infertility clinic due to reproductive problems consistent with the WHO manual from 1999, which were reassessed according to the manual from 2010. Semen results from 571 males from couples undergoing fertility investigation were analyzed. All subjects included in the study had no abnormalities during examination. In 64 samples (11.2%), a leukocyte count above 1 x 106/ml was found and their semen volume (median 3.2 ml) was significantly lower in comparison with the group without leukocytes (3.6 ml; p <0.001). Normal semen parameters were found in 290 subjects (50.8%) according to the 1999 manual and in 362 men (63.4%) according to the 2010 manual. The normozoospermia group, according to the 2010 manual, had a significantly lower percentage of sperm with progressive motility, motile sperm concentration, and total number of motile sperm in comparison with the normozoospermia group according to the manual from 1999. It seems that routine semen analysis is not sufficient to estimate male fertility potential and some men with normal semen parameters may be subfertile. Further investigations are needed.
PMCID: PMC3921700  PMID: 24578858
semen quality; subfertility; sperm concentration; sperm motility
7.  Correlation between seminal lead and cadmium and seminal parameters in idiopathic oligoasthenozoospermic males 
The exact causes of the decline in semen quality are not yet known, environmental factors have been considered to play an important role. Lead (Pb) and Cadmium (Cd) are two of the well-known reproductive toxicants to which humans are exposed occupationally and environmentally and can lead to negative effects on the testicular functions. The aim of this study was to evaluate lead and cadmium levels in seminal plasma of men with idiopathic oligoasthenozoospermia in comparison to fertile healthy controls and to correlate these levels with conventional semen parameters, sperm hypo-osmotic swelling (HOS) percentage, sperm DNA fragmentation percentage, and semen reactive oxygen species (ROS) levels.
Material and Methods
Thirty infertile male patients with idiopathic oligo and/or asthenozoospermia and thirty healthy fertile men, which was the control group, were included in the study. Lead and cadmium levels in seminal plasma, semen parameters, sperm HOS, sperm DNA fragmentation percentage and semen ROS assay were measured in all subjects.
There was a significant increase in seminal lead and cadmium levels among infertile males in comparison to controls. There were significant negative correlations between seminal lead and cadmium levels on one hand and certain semen parameters especially progressive sperm motility and vitality (HOS). Importantly, significant positive correlations were noted between seminal lead and cadmium levels on one hand and sperm DNA fragmentation percentage and semen ROS level in infertile men and controls on the other hand.
Thus, men with idiopathic male infertility had higher levels of lead and cadmium in their semen which correlated with impairment of sperm motility and vitality percentages and more importantly with higher sperm DNA fragmentation% and semen ROS level.
PMCID: PMC3921854  PMID: 24579002
azoospermia; lead; cadmium
8.  Environmental exposure to arsenic may reduce human semen quality: associations derived from a Chinese cross-sectional study 
Environmental Health  2012;11:46.
Recent observations in in vitro and in vivo models suggest that arsenic (As) is an endocrine disruptor at environmentally-relevant levels. When exposed to As, male rats and mice show steroidogenic dysfunction that can lead to infertility. However, the possible effects of As on human male semen quality remain obscure.
We monitored the profile of As species in the urine of a reproductive-age human cohort and assessed its association with semen quality. Men (n = 96) were recruited in an infertility clinic from July 2009 to August 2010 in the Affiliated Hospital of Chongqing Institute for Population and Family Planning. Five urinary As species were analyzed by high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Clinical information on the semen volume, sperm concentration and motility was employed to catalogue and evaluate semen quality according to WHO guidelines. As species concentrations in addition to other continuous variables were dichotomized by the medians and modelled as categorical variables in order to explore using the binary logistic regression possible associations between As exposure and semen quality.
Urinary concentrations (geometric mean ± SD, μg g-1 creatinine) of different As species were 7.49 (±24.8) for AsB, 20.9 (±13.7) for DMA, 2.77 (±3.33) for MMA, and 4.03 (±3.67) for Asi (AsiIII and AsiV). DMA concentrations above the median were significantly associated with below-reference sperm concentrations (P =0.02) after adjusting for age, body mass index (BMI), abstinence, smoking and drinking habits. In addition, smoking was positively associated with MMA.
Reduced parameters in human semen quality are positively associated with As exposure in a reproductive-age Chinese cohort.
PMCID: PMC3419631  PMID: 22776062
Arsenic; China; Cross-sectional study; Human reproduction; Semen quality
9.  No Decline in Semen Quality Among Potential Sperm Donors in Sydney, Australia, Between 1983 and 2001 
Purpose: To determine whether the quality of semen has changed over time in men screened for semen donation.
Methods: All 448 men volunteering for semen donation between 1983 and 2001 at a donor insemination clinic in Sydney, Australia, were included in this longitudinal single centre observational analysis of semen parameters. There was no selection for fertility or marital status but all volunteers had to be aged between 18 and 40 years.
Results: There was no change in the total sperm count during the study period (r = 0.065, P = 0.17) using a linear regression model. The ejaculate volume did not change (r = 0.002, P = 0.97), while an increase in sperm motility was seen (Spearman R = 0.194, P < 0.001).
Conclusion: The semen quality of volunteers for sperm donation presenting to our donor insemination clinic in Sydney between 1983 and 2001 has not declined.
PMCID: PMC3455220  PMID: 12166634
Semen donation; semen quality; sperm count; sperm motility
10.  Sperm Chromatin Integrity: Etiologies and Mechanisms of Abnormality, Assays, Clinical Importance, Preventing and Repairing Damage 
The standard semen analysis is the first line and the most popular laboratory test in the diagnosis of male fertility. It evaluates sperm concentration, motility, morphology and their vitality. However, it is well-known that normal results of semen analysis can not exclude men from the causes of couples′ infertility. One of the most important parameters of sperm in its fertilizing potential is “Sperm chromatin integrity” that has direct positive correlation with Assisted Reproductive Techniques (ART) outcomes including; fertilization rate, embryo quality, pregnancy and successful delivery rate. It seems that sperm DNA chromatin integrity provides better diagnostic and prognostic approaches than standard semen parameters. For these reasons under-standing the sperm chromatin structure, etiology of sperm chromatin abnormality, identification factors that disturbs sperm chromatin integrity and the mechanism of their action can help in recognizing the causes of couples′ infertility. Various methods of its evaluation, its importance in male fertility, clinical relevance in the outcomes of ART and application of laboratory and medical protocols to improve this integrity have valuable position in diagnosis and treatment of male infertility. There has recently been interest in the subject and its application in the field of andrology. Therefore, with regard to the above mentioned importance of sperm chromatin integrity, this review article describes details of the useful information pertaining to sperm DNA damage including the origins, assessments, etiologies, clinical aspects, and prevention of it.
PMCID: PMC3558136  PMID: 23408441
Chromatin; DNA Damage; Fertility; Male; Spermatozoa
11.  Semen quality in relation to biomarkers of pesticide exposure. 
Environmental Health Perspectives  2003;111(12):1478-1484.
We previously reported reduced sperm concentration and motility in fertile men in a U.S. agrarian area (Columbia, MO) relative to men from U.S. urban centers (Minneapolis, MN; Los Angeles, CA; New York, NY). In the present study we address the hypothesis that pesticides currently used in agriculture in the Midwest contributed to these differences in semen quality. We selected men in whom all semen parameters (concentration, percentage sperm with normal morphology, and percentage motile sperm) were low (cases) and men in whom all semen parameters were within normal limits (controls) within Missouri and Minnesota (sample sizes of 50 and 36, respectively) and measured metabolites of eight current-use pesticides in urine samples provided at the time of semen collection. All pesticide analyses were conducted blind with respect to center and case-control status. Pesticide metabolite levels were elevated in Missouri cases, compared with controls, for the herbicides alachlor and atrazine and for the insecticide diazinon [2-isopropoxy-4-methyl-pyrimidinol (IMPY)]; for Wilcoxon rank test, p = 0.0007, 0.012, and 0.0004 for alachlor, atrazine, and IMPY, respectively. Men from Missouri with high levels of alachlor or IMPY were significantly more likely to be cases than were men with low levels [odds ratios (ORs) = 30.0 and 16.7 for alachlor and IMPY, respectively], as were men with atrazine levels higher than the limit of detection (OR = 11.3). The herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and metolachlor were also associated with poor semen quality in some analyses, whereas acetochlor levels were lower in cases than in controls (p = 0.04). No significant associations were seen for any pesticides within Minnesota, where levels of agricultural pesticides were low, or for the insect repellent DEET (N,N-diethyl-m-toluamide) or the malathion metabolite malathion dicarboxylic acid. These associations between current-use pesticides and reduced semen quality suggest that agricultural chemicals may have contributed to the reduction in semen quality in fertile men from mid-Missouri we reported previously.
PMCID: PMC1241650  PMID: 12948887
12.  Decline of semen quality and increase of leukocytes with cigarette smoking in infertile men 
Background: Previous researches about the effect of smoking on semen quality are contradictory, and the mechanism behind the harmful effect of smoking on semen quality still remains unclear until today.
Objective: The objectives of this study are evaluation of the relationship between smoking and fertility, investigation of the effects of cigarette smoking on sperm parameters and detection of presence of leukocytes within the semen of idiopathic infertile men from Northeastern China.
Materials and Methods: A retrospective study of 1512 infertile patients who visited affiliated hospitals of Jilin University from 2007-2010 were enrolled in this study. Patients were assigned into one non-smoking and one smoking group which was divided into mild, moderate and heavy subgroups. Sperm parameters (including leukocytes) and sperm morphology analysis were performed using standard techniques.
Results: Compared with non-smokers, smokers had a significant decrease in semen volumes (p=0.006), rapid progressive motility (p=0.002) and sperm viability (p=0.019); moreover, smokers had a significant increase in the levels of immotile sperms (p=0.005) and semen leukocytes (p=0.002); pH and sperm concentration were not statistically significant (p=0.789 and p=0.297 respectively). Sperm motion parameters were all lower in the smokers except for beat-cross frequency (Hz) (BCF). Further, the percentage of normal morphology sperm was decreased significantly in smokers (p=0.003), the sperm morphology was worse with increasing degree of smoking.
Conclusion: These findings suggest that smoking leads to a significant decline in semen quality and higher levels of leukocytes, thus smoking may affects the fertilization efficiency.
PMCID: PMC3941345  PMID: 24639795
Smoking; Male infertility; Semen analysis; Leukocyte
13.  The Correlation of Sperm Chromatin Decondensation Following In Vitro Exposure to Heparin and Sperm Penetration Rates 
Purpose:The aim of this study was to evaluate the possible correlation of low-dose heparin-induced decondensation of sperm chromatin with sperm concentration, motility, morphology, membrane hypoosmotic response, ejaculate volume, and the ability of sperm to penetrate zona-free hamster oocytes.
Methods:Twenty-two donors of known fertility and 105 patients undergoing evaluation at an andrology laboratory were evaluated by standard World Health Organization semen analysis techniques and a modified sperm penetration assay (SPA). An aliquot was also incubated for 60 min and Ham's F10 medium containing 50 USP/ml heparin. The percentage of sperm undergoing chromatin decondensation was evaluated and correlated to SPA rates and semen quality parameters.
Results:No significant correlation was observed between semen parameters and decondensation rates. A nonsignificant (P = 0.11) inverse correlation (P = −0.21) wax observed between SPA rates and chromatin decondensation. Significant (P < 0.001) differences were observed in the decondensation rate of donors (3.7 ± 0.6), patients with normal SPA rates (7.8 ± 1.5), and patients with decreased SPA rates (21.7 ± 1.8). The decondensation rates were significantly different (P < 0.01) between patients with a normal SPA rate and patients with a decreased SPA rate.
Conclusions:These data indicate a significant inverse relationship between the SPA rate, which has previously been shown to correlate highly with fertilization ability, and heparin-induced sperm chromatin decondensation.
PMCID: PMC3454926  PMID: 9822985
protamine; chromatin; decondensation; capacitation
14.  Sperm transcriptome profiling in oligozoospermia 
Investigate in what extent sperm transcriptome of infertile men is different from that of fertile individuals.
Semen samples were collected for determination of sperm parameters as well as for RNA isolation. Gene expression profile was investigated in spermatozoa of 8 infertile and 3 fertile men by microarray analysis using the Affymetrix Chip HG-U133 Plus 2.0.
We observed up to 33-fold reduction expression of genes involved in spermatogenesis and sperm motility. Furthermore, there is an important decrease in expression of genes involved in DNA repair as well as oxidative stress regulation. In this study, we also show a striking drop in expression of histone modification genes.
We found that transcription profile in germ cells of men with idiopathic infertility is different from that of fertile individuals. Interestingly, about 15% of the regulated genes (Eddy Rev Reprod 4:23–30, 1999) play a role in spermatogenesis.
PMCID: PMC3252406  PMID: 21989496
Sperm; Transcripts; Oligoasthenoteratozoospermia; Male infertility; Microarray
15.  The Relationship of Urinary Metabolites of Carbaryl/Naphthalene and Chlorpyrifos with Human Semen Quality 
Environmental Health Perspectives  2004;112(17):1665-1670.
Most of the general population is exposed to carbaryl and other contemporary-use insecticides at low levels. Studies of laboratory animals, in addition to limited human data, show an association between carbaryl exposure and decreased semen quality. In the present study we explored whether environmental exposures to 1-naphthol (1N), a metabolite of carbaryl and naphthalene, and 3,5,6-trichloro-2-pyridinol (TCPY), a metabolite of chlorpyrifos and chlorpyrifos-methyl, are associated with decreased semen quality in humans. Subjects (n = 272) were recruited through a Massachusetts infertility clinic. Individual exposures were measured as spot urinary concentrations of 1N and TCPY adjusted using specific gravity. Semen quality was assessed as sperm concentration, percent motile sperm, and percent sperm with normal morphology, along with sperm motion parameters (straight-line velocity, curvilinear velocity, and linearity). Median TCPY and 1N concentrations were 3.22 and 3.19 μg/L, respectively. For increasing 1N tertiles, adjusted odds ratios (ORs) were significantly elevated for below-reference sperm concentration (OR for low, medium, and high tertiles = 1.0, 4.2, 4.2, respectively; p-value for trend = 0.01) and percent motile sperm (1.0, 2.5, 2.4; p-value for trend = 0.01). The sperm motion parameter most strongly associated with 1N was straight-line velocity. There were suggestive, borderline-significant associations for TCPY with sperm concentration and motility, whereas sperm morphology was weakly and nonsignificantly associated with both TCPY and 1N. The observed associations between altered semen quality and 1N are consistent with previous studies of carbaryl exposure, although suggestive associations with TCPY are difficult to interpret because human and animal data are currently limited.
PMCID: PMC1253656  PMID: 15579410
biological markers; environment; human; pesticides; semen
16.  Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles 
Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval.
Two semen samples were obtained from 240 men from an unselected group of couples undergoing infertility investigation and treatment. Mean time interval between the two semen evaluations was 119 +/- 102 days. No clinical or surgical treatment was realized between the two observations. Spermatozoa were analyzed at greater than or equal to 8400× magnification by inverted microscope equipped with DIC/Nomarski differential interference contrast optics. At least 200 motile spermatozoa per semen sample were evaluated and percentages of normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV/one or more vacuoles occupying >50% of the sperm nuclear area) were determined. A spermatozoon was classified as morphologically normal when it exhibited a normal nucleus (smooth, symmetric and oval nucleus, width 3.28 +/- 0.20 μm, length 4.75 +/- 0.20 μm/absence of vacuoles occupying >4% of nuclear area) as well as acrosome, post-acrosomal lamina, neck and tail, besides not presenting cytoplasm around the head. One examiner, blinded to subject identity, performed the entire study.
Mean percentages of morphologically normal and LNV spermatozoa were identical in the two MSOME analyses (1.6 +/- 2.2% vs. 1.6 +/- 2.1% P = 0.83 and 25.2 +/- 19.2% vs. 26.1 +/- 19.0% P = 0.31, respectively). Regression analysis between the two samples revealed significant positive correlation for morphologically normal and for LNV spermatozoa (r = 0.57 95% CI:0.47-0.65 P < 0.0001 and r = 0.50 95% CI:0.38-0.58 P < 0.0001, respectively).
The significant positive correlation and absence of differences between two sperm samples evaluated after a time interval with respect to normal morphology and LNV spermatozoa indicated that MSOME seems reliable (at least for these two specific sperm forms) for analyzing semen. The present result supports the future use of MSOME as a routine method for semen analysis.
PMCID: PMC2889997  PMID: 20529256
17.  Immature germ cells in semen – correlation with total sperm count and sperm motility 
Current data regarding infertility suggests that male factor contributes up to 30% of the total cases of infertility. Semen analysis reveals the presence of spermatozoa as well as a number of non-sperm cells, presently being mentioned in routine semen report as “round cells” without further differentiating them into leucocytes or immature germ cells.
The aim of this work was to study a simple, cost-effective, and convenient method for differentiating the round cells in semen into immature germ cells and leucocytes and correlating them with total sperm counts and motility.
Materials and Methods:
Semen samples from 120 males, who had come for investigation for infertility, were collected, semen parameters recorded, and stained smears studied for different round cells. Statistical analysis of the data was done to correlate total sperm counts and sperm motility with the occurrence of immature germ cells and leucocytes. The average shedding of immature germ cells in different groups with normal and low sperm counts was compared. The clinical significance of “round cells” in semen and their differentiation into leucocytes and immature germ cells are discussed.
Round cells in semen can be differentiated into immature germ cells and leucocytes using simple staining methods. The differential counts mentioned in a semen report give valuable and clinically relevant information. In this study, we observed a negative correlation between total count and immature germ cells, as well as sperm motility and shedding of immature germ cells. The latter was statistically significant with a P value 0.000.
PMCID: PMC3793356  PMID: 24130411
Immature germ cells; round cells; semen; total sperm count
18.  The proportion of beef bulls in western Canada with mature spermiograms at 11 to 15 months of age. 
The Canadian Veterinary Journal  2001;42(10):783-787.
A study was conducted to determine the proportion of yearling beef bulls that have mature spermiograms at 11 to 15 months of age, and to evaluate the relationship between semen quality traits, age, and scrotal circumference. Semen samples and data on sperm motility and scrotal circumference measurements were obtained from 1641 bulls of 14 breeds. Criteria for a satisfactory spermiogram included sperm concentration > or = 400 x 10(6)/mL, > or = 60% progressively motile sperm, and > or = 70% morphologically normal sperm. The mean scrotal circumference measurements for all bulls combined were 33.4, 34.4, 35.2, 35.8, and 35.3 cm at 11, 12, 13, 14, and 15 months of age, respectively. The percentage of bulls with matures spermiograms at 11, 12, 13, 14, and 15 months of age were 20%, 30%, 51%, 52%, and 61%, respectively. There was a high positive correlation (r = 0.9) between the number of bulls with > or = 70% normal sperm and scrotal circumference measurement. The main types of morphologic defects observed in immature bulls were proximal droplets and midpiece defects.
PMCID: PMC1476580  PMID: 11665426
19.  Assessment of Chromatin Maturity in Human Spermatozoa: Useful Aniline Blue Assay for Routine Diagnosis of Male Infertility 
Advances in Urology  2013;2013:578631.
During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 (n = 31): immature sperm <20% and G2 (n = 20): immature sperm ≥20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities (P = 0.01) and microcephalic sperm (P = 0.02). We founded significant correlation between sperm immaturity and acrosome abnormalities (r = 0.292; P = 0.03). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility.
PMCID: PMC3808709  PMID: 24198830
20.  Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen 
Asian Journal of Andrology  2010;12(5):760-765.
Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L−1 (group I, control), 5 mmol L−1 (group II), 10 mmol L−1 (group III) and 15 mmol L−1 (group IV). Semen suspensions were loaded in straws (0.5 mL) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher (P < 0.01) percentage of progressive motility, viability and acrosomal integrity in two L-cysteine-supplemented groups (group II and group III) compared with the control. There was a biphasic effect of L-cysteine, with the highest percentage of progressive motility, viability and acrosomal integrity in group III. In conclusion, 5 or 10 mmol L−1 was the optimum concentration of L-cysteine to be added to the LEY extender for improving the quality of frozen–thawed boar semen.
PMCID: PMC3739316  PMID: 20601963
antioxidant; boar semen; cryopreservation; L-cysteine
21.  Effects of Spinal Cord Injury on Semen Parameters 
Neurogenic reproductive dysfunction in men with spinal cord injury (SCI) is common and the result of a combination of impotence, ejaculatory failure, and abnormal semen characteristics. It is well established that the semen quality of men with SCI is poor and that changes are seen as early as 2 weeks after injury. The distinguishing characters of poor quality are abnormal sperm motility and viability. In the majority of the men with SCI, the sperm count is not abnormal. We elaborate on the effects of the SCI on semen parameters that may contribute to poor motility and poor viability.
PubMed and MEDLINE databases were searched using the following key words: spinal cord injuries, fertility, sexual dysfunction, and spermatogenesis. All literature was reviewed by the team of authors according to the various stages of sperm development and transport in the male reproductive cycle.
The cause of asthenozoospermia appears to be multifactorial.
Current literature does not support the preeminence of a single factor relating to neurogenic reproductive dysfunction in men with SCI. After SCI, there is ample evidence of disturbance of sperm production, maturation and storage, and transport due to an abnormal neuroendocrine milieu. Semen quality seems to be primarily affected by changes to the seminal plasma constituents, type of bladder management, and the neurogenic impairment to the ejaculatory function. Further focused and structured studies are required.
PMCID: PMC2435039  PMID: 18533408
Spinal cord injuries; Infertility, male; Semen quality; Reproductive medicine; Paraplegia; Tetraplegia; Electroejaculation; Vibroejaculation
22.  Exposure to Environmental Ozone Alters Semen Quality 
Environmental Health Perspectives  2005;114(3):360-365.
Idiopathic male infertility may be due to exposure to environmental toxicants that alter spermatogenesis or sperm function. We studied the relationship between air pollutant levels and semen quality over a 2-year period in Los Angeles, California, by analyzing repeated semen samples collected by sperm donors. Semen analysis data derived from 5,134 semen samples from a sperm donor bank were correlated with air pollutant levels (ozone, nitrogen dioxide, carbon monoxide, and particulate matter < 10 μm in aerodynamic diameter) measured 0–9, 10–14, and 70–90 days before semen collection dates in Los Angeles between January 1996 and December 1998. A linear mixed-effects model was used to model average sperm concentration and total motile sperm count for the donation from each subject. Changes were analyzed in relationship to biologically relevant time points during spermatogenesis, 0–9, 10–14, and 70–90 days before the day of semen collection. We estimated temperature and seasonality effects after adjusting for a base model, which included donor’s date of birth and age at donation. Forty-eight donors from Los Angeles were included as subjects. Donors were included if they collected repeated semen samples over a 12-month period between January 1996 and December 1998. There was a significant negative correlation between ozone levels at 0–9, 10–14, and 70–90 days before donation and average sperm concentration, which was maintained after correction for donor’s birth date, age at donation, temperature, and seasonality (p < 0.01). No other pollutant measures were significantly associated with sperm quality outcomes. Exposure to ambient ozone levels adversely affects semen quality.
PMCID: PMC1392229  PMID: 16507458
air pollution; epididymis; male infertility; ozone; semen analysis; sperm concentration; total motile sperm count
23.  The effects of male aging on semen quality, sperm DNA fragmentation and chromosomal abnormalities in an infertile population 
To investigate the effects of male aging on semen quality, DNA fragmentation and chromosomal abnormalities in the spermatozoa of infertile patients and fertile men.
Semen samples of 140 infertile patients (24–76 years) and 50 men with proven fertility (25–65 years) were analyzed according to WHO guidelines. DNA fragmentation was detected by TUNEL assay, while aneuploidy was assessed by FISH.
In the patient group, semen volume and vitality of spermatozoa decreased significantly with age, while sperm concentration showed a statistically significant increase with age. DNA fragmentation as well as disomy of sex chromosomes and disomy 8 did not show a statistically significant change with age. However, the diploidy rate was significantly increased with patient’s age. In the control group, conventional semen parameters as well as DNA fragmentation and chromosomal abnormalities did not show a statistically significant with age.
Increased age in infertile men is associated with an increase in sperm concentration and diploidy, as well as a decline in semen volume and sperm vitality. However motility, morphology and DNA fragmentation are not affected by male age.
PMCID: PMC3151353  PMID: 21287403
Aneuploidy; DNA fragmentation; Male infertility; Paternal age; Semen parameters
24.  Effects of Varicocelectomy on Anti-sperm Antibody in Patients with Varicocele 
Anti-sperm antibody (ASA) can decrease sperm motility and, therefore, it is a cause of male infertility. The aim of this study was to evaluate the effects of varicocelectomy on anti-sperm antibody in patients with varicocele.
This observational study was conducted on 90 patients with varicocele at Sina and Imam Khomeini hospitals during 2006 to 2009. All varicocelectomy candidates were selected for ASA assessment both in semen and serum before and after surgery. ASA level was measured using a direct method for semen and an indirect method of Sperm MAR test, for serum. Paired t-test and McNemar's test were used for data analysis, and p<0.05 was considered statistically significant.
ASA level in semen was 13.7% before, and 15.7% after three month of varicocelectomy (p=0.881). Serum level of ASA before and after surgery were 13.6% and 21.7%, respectively (p=0.033). Three parameters including sperm count, motility and morphology showed recovery following, varicocelectomy, but only the difference in sperm motility was significant (p<0.05).
This study showed that varicocelectomy has no effect on semen ASA. Although serum antibody has been shown to increase after varicocelectomy but sperm motility will improve. Varicocelectomy seems to have a beneficial effect on semen parameters in infertile men with varicocele.
PMCID: PMC3719315  PMID: 23926568
Anti-sperm Antibody; Infertility; Sperm Motility; Varicocelectomy
25.  Declining semen quality among south Indian infertile men: A retrospective study 
Male reproductive function has recently attracted increasing attention due to reports on time-related decline in semen quality. Furthermore, regional differences in the semen quality have also been reported.
To investigate the semen quality among large cohort of infertile individuals at a regional level, in terms of the sperm concentration, total sperm motility, sperm morphology and incidence of azoospermia over a period of 13 years.
University infertility clinic at Kasturba Hospital, Manipal which is a tertiary healthcare centre serving the general population.
Retrospective analysis.
This includes a total of 7770 subjects who presented for semen analysis from 1993 to 2005. The data regarding ejaculate volume, sperm density, motility, morphology and the incidence of azoospermia were collected.
One way analysis of variance (ANOVA), regression analysis and Chi square analysis.
The average sperm density among infertile men during 2004-2005 was 26.61 ± 0.71 millions/mL which was significantly lower than the average sperm density observed in 1993-1994 (38.18 ± 1.46 millions/mL). Similar trend was also observed for sperm motility (47.14% motile sperms vs. 61.16%) and normal sperm morphology (19.75% vs. 40.51%). Interestingly, the incidence of severe oligospermia (mean sperm density <10 millions/mL) observed in 2002-2005 and 1993-1997 demonstrated a significant inverse relationship (P < 0.001).
Our study provides the first evidence that the quality of human semen evaluated for infertility is deteriorating in the southern part of the India over the years, probably due to environmental, nutritional, life style or socioeconomic causes.
PMCID: PMC2700673  PMID: 19562058
Azoospermia; infertility; reproductive function; semen quality; seminal parameters; sperm concentration; sperm decline; spermatozoa

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