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1.  Human semen quality in the new millennium: a prospective cross-sectional population-based study of 4867 men 
BMJ Open  2012;2(4):e000990.
Considerable interest and controversy over a possible decline in semen quality during the 20th century raised concern that semen quality could have reached a critically low level where it might affect human reproduction. The authors therefore initiated a study to assess reproductive health in men from the general population and to monitor changes in semen quality over time.
Cross-sectional study of men from the general Danish population. Inclusion criteria were place of residence in the Copenhagen area, and both the man and his mother being born and raised in Denmark. Men with severe or chronic diseases were not included.
Danish one-centre study.
4867 men, median age 19 years, included from 1996 to 2010.
Outcome measures
Semen volume, sperm concentration, total sperm count, sperm motility and sperm morphology.
Only 23% of participants had optimal sperm concentration and sperm morphology. Comparing with historic data of men attending a Copenhagen infertility clinic in the 1940s and men who recently became fathers, these two groups had significantly better semen quality than our study group from the general population. Over the 15 years, median sperm concentration increased from 43 to 48 million/ml (p=0.02) and total sperm count from 132 to 151 million (p=0.001). The median percentage of motile spermatozoa and abnormal spermatozoa were 68% and 93%, and did not change during the study period.
This large prospective study of semen quality among young men of the general population showed an increasing trend in sperm concentration and total sperm count. However, only one in four men had optimal semen quality. In addition, one in four will most likely face a prolonged waiting time to pregnancy if they in the future want to father a child and another 15% are at risk of the need of fertility treatment. Thus, reduced semen quality seems so frequent that it may impair the fertility rates and further increase the demand for assisted reproduction.
Article summary
Article focus
A paper by Carlsen et al 20 years ago (BMJ 1992;305:609–13) raised controversy with evidence of a decline in semen quality, and several studies on semen quality in human populations have followed.
There has been a lack of larger, prospectively collected quality-controlled data on semen quality in the general population.
Key messages
This study brings good and bad news.
Fifteen years monitoring of semen quality in men of the general population indicated a slight increase in both median sperm concentration and total sperm count.
However, still only a fraction of the men (23%) had optimal sperm concentration and sperm morphology, and the median percentage of abnormal spermatozoa was as high as 93% with no sign of improvement during the study period.
Approximately 15% of the men had a sperm concentration at a level that would indicate a high risk of needing future fertility treatment if they want to father a child, and another 27% of the men will be at risk of a prolonged waiting time to pregnancy.
Strengths and limitations of this study
Large prospective study of semen quality among men of the general population unselected with regard to fertility.
Standardised inclusion and investigation procedures.
Lack of historical, directly comparable data.
PMCID: PMC3391374  PMID: 22761286
2.  Decline in semen concentration and morphology in a sample of 26 609 men close to general population between 1989 and 2005 in France 
Are temporal trends and values of semen quality parameters in France identifiable in partners of totally infertile women?
Among a sample of 26 609 partners of totally infertile women undergoing an assisted reproductive technology (ART) procedures in the whole of France over a 17-year period, there was a continuous decrease in semen concentration of about 1.9% per year and a significant decrease in the percentage with morphologically normal forms but no global trend for motility.
A global decrease in human sperm quality is still debated as geographical differences have been shown, and many criticisms have risen concerning studies with small and biased study populations or inappropriate statistical methodology. However, growing biological, toxicological, experimental and human exposure data support the endocrine disruptors' hypothesis assuming that fetal exposure to endocrine disruptors could impair reproductive outcomes.
This was a retrospective and descriptive study using data registered by Fivnat, the professional association in charge of statistics for ART in France during the 1989–2005 study period. Data were provided by 126 main ART centres over the whole metropolitan territory. The source population included 154 712 men, aged 18–70, who were partners of couples undergoing their first ART cycle and for whom semen quality indicators (concentration, total motility and percentage of morphologically normal forms), measured on fresh ejaculated semen, were available.
The study population was 26 609 partners of women who had both tubes either absent or blocked. The temporal trends for each indicator of semen quality were modelled using a generalized additive model that allowed for nonlinear relationships between variables and were adjusted for season and age. In-depth sensitivity analyses included the reiteration of the analysis on data from a second spermiogram available for each man and on another subsample of men diagnosed as fertile. Variables such as centre, technique (standard in vitro fertilization or intra-cytoplasmic sperm injection) and an interaction factor between technique and time were also included in the model.
There was a significant and continuous decrease in sperm concentration of 32.2% [26.3–36.3] during the study period. Projections indicate that concentration for a 35-year-old man went from an average of 73.6 million/ml [69.0–78.4] in 1989 to 49.9 million/ml [43.5–54.7] in 2005. A significant, but not quantifiable, decrease in the percentage of sperm with morphologically normal forms along the 17-year period was also observed. There was no global trend but a slight, significant increase in total motility between 1994 and 1998 was observed. The results were robust after sensitivity analysis.
Socioeconomic status could not be controlled for. Despite universal access to medical services in France, couples undergoing ART are expected to have a higher educational level on average compared with those of the general population. Therefore, the real values in the general population could be slightly lower than those presented and the decrease possibly stronger, as the population study is less likely to smoke or be overweight, two factors known to impair semen quality.
As the men were selected without a priori knowledge regarding their semen quality characteristics, the results are expected to be close to the values in the general French population. The very large sample size and the robustness of the results confer great statistical power and credibility to the results. To our knowledge, it is the first study concluding a severe and general decrease in sperm concentration and morphology at the scale of a whole country over a substantial period. This constitutes a serious public health warning. The link with the environment particularly needs to be determined.
No specific funding was sought for this study. The authors have no conflict of interest to declare.
The study has been authorized by the Commission Nationale de l'Informatique et des Libertés (CNIL), the national authority for the protection of personal data collected on individuals (authorization no DE-2010-063 dated 08/09/2010).
PMCID: PMC4042534  PMID: 23213178
epidemiology; sperm quality; environmental effects; male infertility
3.  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
4.  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
5.  Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium infections and semen quality of infertile men 
Genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and mycoplasmas (Mycoplasma genitalium and Mycoplasma hominis) are potentially pathogenic species playing an etiologic role in both genital infections and male infertility. Reports are, however, controversial regarding the effects of these microorganisms infections in the sperm seminological variables. This study aimed at determining the frequency of genital ureplasmas and mycoplasmas in semen specimens collected from infertile men, and at comparing the seminological variables of semen from infected and non-infected men with these microorganisms.
A total of 120 semen samples collected from infertile men were investigated. Semen specimens were examined by in-house PCR-microtiter plate hybridization assay for the presence of genital ureaplasmas and mycoplasmas DNA. Semen analysis was assessed according to the guidelines of the World Health Organization. Standard parametric techniques (t-tests) and nonparametric techniques (Wilcoxon tests) were used for statistical analysis.
The frequency of genital ureaplasmas and mycoplasmas detected in semen samples of infertile men was respectively 19.2% (23/120) and 15.8% (19/120). The frequency of Ureaplasma urealyticum (15%) was higher than that of Mycoplasma hominis (10.8%), Ureaplasma parvum (4.2%) and Mycoplasma genitalium (5%). Mixed species of mycoplasmas and ureaplasmas were detected in 6.7% of semen samples.
Comparison of the parameters of the standard semen analysis between the male partners of the infertile couples with and without genital ureaplasmas and mycoplasmas infection showed that the presence of Mycoplasma hominis DNA in semen samples is associated with low sperm concentration (p = 0.007) and abnormal sperm morphology (p = 0.03) and a negative correlation between sperm concentration and the detection of Mycoplasma genitalium in semen samples of infertile men (p = 0.05). The mean values of seminal volume, pH, vitality, motility and leukocyte count were not significantly related either to the detection of genital mycoplasmas DNA or to the detection of ureaplasmas DNA in semen specimens.
Our results demonstrate that genital mycoplasmas and ureaplasmas seem to be widespread among the male partners of infertile couples in Tunisia. Genital mycoplasmas infections of the male genital tract could negatively influence semen quality. Our results also indicate that PCR-microtiter plate hybridization assay method provides a rapid and effective technique to detect human genital mycoplasmas and ureaplasmas which is useful for etiological and epidemiological studies of these pathogens.
PMCID: PMC2194714  PMID: 17988404
6.  Alterations of the FSH and LH receptor genes and evaluation of sperm ultrastructure in men with idiopathic hypergonadotropic hypogonadism 
Gonadotropins, interacting with their gonadal receptors, play a key role in sexual development, reproductive functions and metabolism. In this study we performed the genetic analysis of FSHR and LHR and semen investigation in 14 infertile men with normal level of T and elevated levels of FSH and/or LH in the absence of other causes of infertility.
Sperm parameters were analysed following WHO (2010) guidelines and sperm morphology by Transmission Electron Microscopy (TEM) analysis mathematically elaborated. FSHR and LHR gene mutations have been searched by PCR technique, followed by DHPLC analysis and direct sequencing.
In FSHR, we found no difference in the frequency between Ala or Thr at position 307, Ser was at codon 680 in all subjects. Three patients had an heterozygous mutation at codon 419. Three intronic polymorphisms (rs2091787, rs6708637, rs1922464) were significantly found compared to controls; the single allele frequency and the odds ratio were calculated. Two new variants: the Cys338Arg and the Gln123Glu were detected in two different patients. Regarding LHR, three patients were heterozygous for the known variant Glu354Lys and two for Ile374Thr. Intronic polymorphisms were not identified. A new variant, the Val144Ile was found. By the routine semen analysis, variable seminal conditions in this group of patients was observed, on the contrary TEM data mathematically elaborated showed a homogeneous decrease in fertility index and increase in sperm pathologies such as apoptosis and immaturity.
The obtained results suggest that a deeper examination of spermatozoa, achieved by the use of more powerful tools such as TEM or molecular analysis, are advisable in patients with hypergonadotropic hypogonadism.
PMCID: PMC3800530  PMID: 23884663
FSHR; LHR; Male infertility; Sperm evaluation; Transmission electron microscopy
7.  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
8.  Semen Parameters in Fertile US Men: The Study for Future Families 
Andrology  2013;1(6):10.1111/j.2047-2927.2013.00125.x.
Establishing reference norms for semen parameters in fertile men is important for accurate assessment, counseling and treatment of men with male factor infertility. Identifying temporal or geographic variability in semen quality also requires accurate measurement of semen parameters in well-characterized, defined populations of men. The Study for Future Families (SFF) recruited men who were partners of pregnant women attending prenatal clinics in Los Angeles CA, Minneapolis MN, Columbia MO, New York City NY and Iowa City IA. Semen samples were collected on site from 763 men (73% White, 15% Hispanic/Latino, 7% Black and 5% Asian or other ethnic group) using strict quality control and well-defined protocols. Semen volume (by weight), sperm concentration (hemacytometer) and sperm motility were measured at each center. Sperm morphology (both WHO, 1999 strict and WHO, 1987) was determined at a central laboratory. Mean abstinence was 3.2 days. Mean (median; 5th – 95th percentile) values were: semen volume, 3.9 (3.7; 1.5 – 6.8) ml; sperm concentration, 60 (67; 12–192) × 106/ml; total sperm count 209 (240; 32–763) × 106; % motile, 51 (52; 28–67) %; and total motile sperm count, 104 (128; 14–395) × 106, respectively. Values for sperm morphology were 11 (10; 3–20) % and 57 (59; 38–72) % normal forms for WHO, 1999 (strict) and WHO, 1987 criteria, respectively. Black men had significantly lower semen volume, sperm concentration and total motile sperm counts than White and Hispanic/Latino men. Semen parameters were marginally higher in men who achieved pregnancy more quickly but differences were small and not statistically significant. The SFF provides robust estimates of semen parameters in fertile men living in five different geographic locations in the US. Fertile men display wide variation in all of the semen parameters traditionally used to assess fertility potential.
PMCID: PMC3812375  PMID: 24009155
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.  Clinical and Structural Features of Sperm Head Vacuoles in Men Included in the In Vitro Fertilization Programme 
BioMed Research International  2014;2014:927841.
The human sperm head vacuoles and their role in male infertility are still poorly understood. The aim of this study was to identify the clinical and ultrastructural features of human sperm head vacuoles in men included in the in vitro fertilization programme: men with normal (normozoospermia) and impaired sperm morphology (teratozoospermia). The sperm samples were observed under 6000-time magnification using motile sperm organelle morphology examination (MSOME). The proportion of sperm with head vacuoles was evaluated and related to the outcome of in vitro fertilization. The sperm of men with impaired sperm morphology was characterized by a higher proportion of sperm head vacuoles. The sperm head vacuoles were related to impaired semen quality (sperm concentration, motility, and morphology) but were not influenced by male factors (semen volume, height, age, weight, or body mass index). Moreover, sperm head vacuoles were related to impaired fertilization rate merely after classical in vitro fertilization (IVF), while there was no relation to pregnancy. In a subgroup of men, the sperm was fixed and observed by transmission electron microscopy (TEM). The ultrastructural study revealed that sperm head vacuoles are large nuclear indentations of various sizes and positions, packed with membranous material organized in membrane whorls (MW).
PMCID: PMC4000983  PMID: 24818161
11.  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
12.  Paternal age and assisted reproductive outcomes in ICSI donor oocytes: is there an effect of older fathers? 
Human Reproduction (Oxford, England)  2014;29(10):2114-2122.
Does paternal age affect semen quality and reproductive outcomes in oocyte donor cycles with ICSI?
Paternal age is associated with a decrease in sperm quality, however it does not affect either pregnancy or live birth rates in reproductive treatments when the oocytes come from donors <36 years old and ICSI is used.
The weight of evidence suggest that paternal age is associated with decreasing sperm quality, but uncertainty remains as to whether reproductive outcomes are affected. Although developed to treat severe sperm factor infertility, ICSI is gaining popularity and is often used even in the presence of mild male factor infertility.
A retrospective cohort study spanning the period between February 2007 and June 2010. A total of 4887 oocyte donation cycles were included.
Fertilization was carried out by ICSI in all cycles included, and the semen sample used was from the male partner in all cases. The association of male age with semen parameters (volume, concentration, percentage of motile spermatozoa) was analyzed by multiple analysis of covariance. The association of male age with reproductive outcomes (biochemical pregnancy, miscarriage, ongoing pregnancy and live birth rate) was modeled by logistic regression, where the following covariates were introduced: donor age, recipient age, semen state (fresh versus frozen) and number of transferred embryos (3 and 2 versus 1).
We identified a significant relationship between paternal age and all sperm parameters analyzed: for every 5 years of age, sperm volume decreases by 0.22 ml (P < 0.001), concentration increases by 3.1 million sperm/ml (P = 0.003) and percentage motile spermatozoa decreases by 1.2% (P < 0.001). No differences were found in reproductive outcomes (biochemical pregnancy, miscarriage, clinical pregnancy, ongoing pregnancy and live birth) among different male age groups.
The use of donor oocytes, while extremely useful in highlighting the role of male age in reproductive outcomes, limits the generalization of our results to a population of young women with older male partners. No data were available on perinatal and obstetrical outcomes of these pregnancies. Most (75%) cycles used frozen/thawed sperm samples which might have introduced a bias owing to loss of viability after thawing. ICSI was performed in all cycles to control for fertilization method; this technique could mask the natural fertilization rate of poorer sperm samples. Furthermore, we did not use stringent ICSI indications; and our data are therefore not generalizable to cases where only severe male factor is considered. However, male patients were of different racial background, thus allowing generalizing our results to a wider patient base.
Our study suggests that paternal age does not affect reproductive outcomes when the oocyte donor is <36 years of age, indicating that ICSI and oocyte quality can jointly overcome the lower reproductive potential of older semen.
This study was supported in part by Fundació Privada EUGIN. The authors have no conflicts of interest to declare.
PMCID: PMC4164148  PMID: 25073975
male age; sperm; donor oocyte; ICSI; pregnancy rate
13.  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
14.  Molecular Detection of Chlamydia trachomatis and Other Sexually Transmitted Bacteria in Semen of Male Partners of Infertile Couples in Tunisia: The Effect on Semen Parameters and Spermatozoa Apoptosis Markers 
PLoS ONE  2014;9(7):e98903.
This study was undertaken to determine the prevalence of Chlamydia trachomatis, Mycoplasmas, and Ureaplasmas in semen samples of the male partners of infertile couples and to investigate whether Chlamydia trachomatis could initiate apoptosis in human spermatozoa. A total of 85 males partners of infertile couples undergoing routine semen analysis according to World Health Organization guidelines were included. Specimens were examined for the presence of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum and Ureaplasma parvum by Real time PCR (qPCR). Semen specimens were analysed for the appearance of apoptotic markers (sperm DNA fragmentation, activated caspase 3 levels, mitochondrial membrane potential (ΔΨm)) using flow cytometry. C. trachomatis, N. gonorrhoeae, U. urealyticum, M genitalium were detected in semen samples of 13 (15.2%), 5 (5.8%), 5 (5.8%) and 3 (3.5%) male partners of infertile couples, respectively. M. hominis and U. parvum were detected in semen sample of only one patient (1.1%). The semen of infertile men positive for C. trachomatis showed lower mean of semen count and lower rapid progressive motility (category [a]) of spermatozoa compared to uninfected men with statistically significances (p = 0.02 and p = 0.04, respectively). Flow cytometry analyses demonstrated a significant increase of the mean rate of semen with low ΔΨm and caspase 3 activation of infertile men positive for C. trachomatis compared to uninfected men (p = 0.006 and p = 0.001, respectively). DNA fragmentation was also increased in sperm of infertile men positive for C. trachomatis compared to uninfected men but without statistical significances (p = 0.62). Chlamydial infection was associated to loss of ΔΨm and caspase 3activation. Thus, C. trachomatis infection could be incriminated in apoptosis induction of spermatozoa. These effects may explain the negative direct impact of C. trachomatis infection on sperm fertilizing ability.
PMCID: PMC4096407  PMID: 25019616
15.  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
16.  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
17.  Heat-Induced Hyperactivation 
Purpose:The objectives of this study were (1) to determine the sperm hyperactivation and related kinematic parameters at 40°C after using four sperm wash procedures and (2) to correlate the heat-induced hyperactivation data with cases of clinical pregnancies from either artificial insemination or standard in vitro fertilization (IVF).
Methods:Semen samples (n = 51) were collected by ejaculation, and semen analyses were carried out to determine the pretreatment data. Sperm kinematic measurements were performed using the Hamilton Thorn HTM-C computer-aided sperm analyzer. Hyperactivation was determined using the sort module on the HTM-C. Membrane integrity was assessed using the hypoosmotic sperm swelling procedure. Sperm morphology and acrosomal status were also determined using the Spermac stain. Each semen specimen was divided and processed through either the swim-up wash, the 1-h test-yolk buffer (TYB) wash, the 1 mg/ml pentoxifylline stimulant procedure, or the two-layer 90:47% gradient colloidal solution procedure. The washed sperm were incubated at 25 or at 40° C for 4 hr. After incubation, kinematic parameters were assessed for the posttreatment data. Semen specimens were obtained on different occasions for artificial insemination or standard IVF. Data from intracytoplasmic sperm injection cases were not included to avoid confounding factors. Live births and/or pregnancies with fetal heartbeat examined by ultrasound were considered clinical pregnancies.
Results:Heat-induced hyperactive motility was significantly higher in sperm of the male partner of pregnant (n = 7) patients compared with nonpregnant (n = 44) patients (mean ± SE, 10.0 ± 3.3 versus 5.5 ± 0.8%) after TYB processing fallowed by 4 hr of incubation at 40°C. This was also observed after colloid (Percoll) processing (11.6 ± 4.6 versus 5.8 ± 0.8%). There were no differences in hyperactivation after 4 hr at 23°C between pregnant and nonpregnant cases. Parameters such as count, volume, motility, viability, and acrosomal status were not different for the groups. However, the percentage of sperm with normal morphology (WHO classification) was twice as high in the pregnant group versus the nonpregnant group.
Conclusions:Heat-induced hyperactivation was associated with fertile sperm and was predictive of pregnancy obtained after artificial insemination or IVF. The association was evident only after TYB or Percoll sperm processing. The study could not confirm the finding of significant decreases in motility after heat treatment of sperm derived from infertile males. The mechanism for heat-induced hyperactivation did not involve membrane integrity or the sperm acrosome, although an involvement of heat shock proteins was postulated. Interestingly, there were no pregnancies when sperm did not exhibit heat-induced hyperactivation.
PMCID: PMC3468205  PMID: 9493064
sperm; computer-aided sperm analyzer (CASA); induction; hyperactivation motility
18.  Relationship between apoptotic markers in semen from fertile men and demographic, hormonal and seminal characteristics 
Asian Journal of Andrology  2012;14(6):890-896.
Apoptosis in the testis has two putative roles during normal spermatogenesis; limitation of the germ cell population to numbers that can be supported by the Sertoli cells, and, possibly, selective depletion of meiotic and postmeiotic abnormal germ cells. We investigated the demographic and biological correlates of the pro-apoptotic marker Fas and the anti-apoptotic marker Bcl-xL in sperm cells of fertile men. Six hundred and four men from Greenland, Poland and Ukraine were consecutively enrolled during their pregnant wife's antenatal visits. Semen analysis was performed as recommended by the World Health Organization. Immunofluorescence coupled to flow cytometry was utilized for detection of apoptotic markers in the sperm cell. DNA damage was assessed by flow cytometry using both the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. The percentage of Fas-positive sperm cells was higher in men with high total sperm count (P<0.01), more motile sperms (P=0.04) and fewer sperm head defects (P=0.05). These associations were consistent within and across study regions. Furthermore, testosterone, follicle-stimulating hormone (FSH) and sexual hormone-binding globulin (SHBG) were significantly negatively correlated with Fas within and across regions as well. The data indicated no association between the anti-apoptotic Bcl-xL marker and semen or personal characteristics. The finding of Fas-positive sperm cells associated with better semen quality in a cohort of spouses of pregnant women seems different from previous data obtained in infertile men and warrants further investigation to clarify the biological significance of sperm apoptotic markers.
PMCID: PMC3720103  PMID: 23064689
apoptosis; Bcl-x protein; Fas-associated death domain protein; fertility; sperm chromatin structure assay (SCSA); spermatozoa; TUNEL assay
19.  Sperm chromosomal aneuploidy and DNA integrity of infertile men with anejaculation 
To explore sperm chromosomal aneuploidy, sperm membrane and DNA integrity in infertile patients with anejaculation.
Semen samples were collected from 18 infertile men with spinal cord injury (SCI) by penile vibratory stimulation (PVS) and from 14 psychogenic anejaculation (PA) patients by percutaneous vasal sperm aspiration (PVSA). These semen samples as well as samples from 16 donors were analyzed using the hypo-osmotic swelling (HOS) test, the sperm chromatin dispersion (SCD) test and multi-color fluorescence in situ hybridization (FISH) with probes specific for chromosomes 13, 18, 21, X and Y.
There were significant differences in the percentages of motile sperm, normal morphologic sperm and sperm DNA fragmentation between the infertile men with SCI and the control group (P < 0.05 and P < 0.01). The sperm motility was significantly greater in the PA-PVSA group than in the SCI-PVS group (P < 0.01). The number of round cells per mL of semen obtained from the 18 SCI patients by PVS was between 1 and 8 million. The rate of sperm DNA fragmentation in the SCI-PVS group was higher than that of the PA-PVSA group (P < 0.05). The aneuploidy rates for the SCI patients were 2.4-fold higher for chromosomes 13, 18 and 21 and 2.2-fold higher for chromosomes X and Y than for patients in the control group (P < 0.0001).
The semen quality is poorer, sperm DNA fragmentation and sperm chromosomal aneuploidies are seen at a higher rate for SCI patients compared to healthy, fertile and normospermic men. Whether the difference in yield is due to increased scrotal temperature, genitourinary infection, or other reasons requires further study.
PMCID: PMC3270129  PMID: 22215471
Sperm chromatin dispersion (SCD) test; FISH; Sperm chromosomal aneuploidy; Anejaculation; Male infertility
20.  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
21.  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
22.  Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men 
BMC Urology  2012;12:6.
Growing evidence indicates that oxidative stress can be a primary cause of male infertility. Non-enzymatic antioxidants play an important protective role against oxidative damages and lipid peroxidation. Human seminal plasma is a natural reservoir of antioxidants. The aim of this study was to determine glutathione (GSH) concentrations, trace element levels (zinc and selenium) and the lipid peroxidation end product, malondialdehyde (MDA), in the seminal plasma of men with different fertility potentials.
Semen samples from 60 fertile men (normozoospermics) and 190 infertile patients (74 asthenozoospermics, 56 oligozoospermics, and 60 teratozoospermics) were analyzed for physical and biochemical parameters. Zinc (Zn) and selenium (Se) levels were estimated by atomic absorption spectrophotometry. Total GSH (GSHt), oxidized GSH (GSSG), reduced GSH (GSHr) and MDA concentrations were measured spectrophotometrically.
Zn and Se concentrations in seminal plasma of normozoospermics were more elevated than the three abnormal groups. Nevertheless, only the Zn showed significant differences. On the other hand, Zn showed positive and significant correlations with sperm motility (P = 0.03, r = 0.29) and count (P < 0.01, r = 0.49); however Se was significantly correlated only with sperm motility (P < 0.01, r = 0.36). GSHt, GSSG and GSHr were significantly higher in normozoospermics than in abnormal groups. We noted a significant association between seminal GSHt and sperm motility (P = 0.03). GSSG was highly correlated to sperm motility (P < 0.001) and negatively associated to abnormal morphology (P < 0.001). GSHr was significantly associated to total sperm motility (P < 0.001) and sperm count (P = 0.01). MDA levels were significantly higher in the three abnormal groups than in normozoospermics. Rates of seminal MDA were negatively associated to sperm motility (P < 0.01; r = -0.24) and sperm concentration (P = 0.003; r = -0.35) Meanwhile, there is a positive correlation between seminal lipid peroxidation and the percentage of abnormal morphology (P = 0.008).
This report revealed that decreased seminal GSH and trace element deficiencies are implicated in low sperm quality and may be an important indirect biomarker of idiopathic male infertility. Our results sustain that the evaluation of seminal antioxidant status in infertile men is necessary and can be helpful in fertility assessment from early stages.
PMCID: PMC3349502  PMID: 22429816
Antioxidants; Idiopathic oligoasthenoteratozoospermia; Male infertility; Oxidative stress; Reactive oxygen species; Spermatozoa; Seminal plasma
23.  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
24.  Correlation between Different Patterns of Hypo-Osmotic Swelling and Sperm Functional Tests 
Sperm membrane integrity is not only important as a barrier between intra- and extra-cellular spaces, but also it can be considered as a sign of DNA integrity. Hypoosmotic swelling test reflects membrane integrity and has been used to evaluate sperm quality. Intracytoplasmic sperm injection (ICSI) in adjunct with hypo-osmotic swelling test (HOST) has been used for treatment of males with asthenozoospermia. Therefore, this study aims to evaluate correlation of different pattern of HOST with sperm parameters, protamine deficiency and apoptosis.
Materials and Methods:
In this case-control study, sixteen semen samples were randomly collected from infertile normozospermic men. Semen samples were divided into two portions as follows: one portion was assessed for sperm parameters according toWorld Health Organization (WHO)-2010, while the other portion, after applying HOST procedure, was used for assessment of sperm morphology, protamine deficiency and late or early apoptosis. Statistical analysis was carried out using the Statistical Package for the Social Studies (SPSS 11.5).
Our results showed that, the lowest odds ratio (OR) of abnormal sperm head morphology and abnormal acrosome was in d-sperm as compared to a-pattern or nonviable spermatozoa (p=0.00, p=0.01). In addition, a significant correlation was observed between d-sperm with sperm concentration and percentage of DNA damage (p=0.03 and p=0.04, respectively). A significant correlation was observed between percentage of sperm motility and DNA fragmentation (r=-0.56; p=0.01). Furthermore, significant correlations were observed between percentages of early apoptotic sperm with protamine deficiency and sperm concentration (p=0.009 and p=0.01, respectively).
Significant correlations exist between d-pattern and sperm DNA integrity. Semen samples with low sperm concentration have low percentage of d-sperm which are mature and intact sperms.
PMCID: PMC3914492  PMID: 24520486
ICSI; HOST; DNA Fragmentation; Protamine
25.  Effect of Dietary Selenium and Vitamin E on Ganders’ Response to Semen Collection and Ejaculate Characteristics 
Biological Trace Element Research  2013;153(1-3):196-204.
Compared to other domestic bird species, geese exhibit the lowest reproductive efficiency (poor semen quality, low egg production, and poor fertility and hatchability rates). From an economic perspective, it is a necessity of improve these reproductive traits. Studies have demonstrated that the essential trace element—selenium—plays key roles in testicular development and the maintenance of spermatogenesis. The aim of the present study was to determine the effect of feed supplementation with organic selenium and vitamin E on ganders’ response to manual semen collection and semen quality. Sixteen 3-year-old White Koluda ganders were randomly divided into two groups. The control group was provided commercial feed while the experimental group was provided with the same commercial feed supplemented with selenium (0.3 mg/kg) and vitamin E (100 mg/kg). The response of individual ganders from both groups to manual semen collection and the quality of the semen collected were evaluated. The supplements increased (P ≤ 0.05) the frequency and decreased the time interval of a complete ejaculatory response of the ganders to manual semen collections (82.7 % supplement vs. 73.5 % control). Males from the supplemented group had significantly higher (P ≤ 0.01; P ≤ 0.05) ejaculate volumes, sperm concentrations, and percentages of viable sperm and lower percentages of immature sperm (spermatids). Lipids peroxidation, expressed in terms of the malondialdehyde concentration, was lower (P ≤ 0.01) in semen of the supplemented group (0.172 nmol/50 × 106) as compared to the controls (0.320 nmol/50 × 106). Moreover, the duration of the reproductive period of the ganders in the experimental group was 1 week longer. The results show that supplemental dietary selenium and vitamin E improved both the ganders’ response to manual semen collection and semen quality. We conclude that such feed supplementation could lead to greater economic benefits through increased reproductive efficiency within the goose production industry.
PMCID: PMC3667365  PMID: 23584843
Organic selenium; Vitamin E; Ganders’ reaction; Semen quality; MDA

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