Computer-assisted Semen Analysis System Research Articles

Fresh semen quality in ejaculates produced by nocturnal emission in men with idiopathic anejaculation

To compare the quality of fresh semen samples collected by nocturnal emission (NE), penile vibratory stimulation (PVS), or electroejaculation (EEJ) from idiopathic anejaculation patients with those collected by masturbation from healthy control subjects. Retrospective clinical study. University medical center. Ninety-one men with idiopathic anejaculation. NE, PVS, EEJ, computer-assisted semen analysis system. Sperm volume, concentration, motility, and morphology. The mean percentage of motile sperm in the NE group was 30.6% ± 3.5% (range 11.2%-50.9%), which is 1.5- and 1.9-fold higher than that of the PVS (20.5% ± 1.3%, range 6.9%-40.2%) and EEJ (16.1% ± 0.9%, range 3.7%-27.3%) groups, respectively. The percentage of sperm that have normal morphology in the NE group was 61.4% ± 4.8%, which is significantly higher than that in the PVS (38.4% ± 2.8%) and EEJ groups (29.5% ± 1.7%), respectively. However, compared with the healthy control group, the sperm motility and normal morphology in the NE, PVS, and EEJ groups were significantly lower. The sperm concentration in the NE group (149.4 ± 16.3 × 10(6) mL(-1)) was similar to that of the PVS group (143.8 ± 13.1 × 10(6) mL(-1)) but was higher than in the EEJ group (96.3 ± 8.6 × 10(6) mL(-1)) and lower than in the control group (225.0 ± 15.0 × 10(6) mL(-1)). Furthermore, the semen volume in the NE group (2.6 ± 0.2 mL) was lower than that of the control group (3.0 ± 0.1 mL). This study is the first to report on the quality of fresh semen obtained by NE in patients with idiopathic anejaculation. Sperm motility and normal morphology were higher in patients who collected sperm by NE than in those who collected sperm by PVS and EEJ.

The effect of nonylphenol on the motility and viability of bovine spermatozoa in vitro

The objective of this in vitro study was to determine the effect of nonylphenol (NP) as an environmental toxicant on the spermatozoa motility and viability. The dose- and time-dependent effect of nonylphenol (1, 10, 100 and 200 μg/mL) dissolved either in 0.1% dimethyl sulfoxide (DMSO) or 0.1% ethanol (ETOH) on the motility and viability of bovine spermatozoa, as a cell model, during several time periods (0 h, 2 h, 4 h and 6 h) were examined. The motility of spermatozoa was determined by the Sperm VisionTM CASA (Computer Assisted Semen Analyzer) system. The results showed decreased spermatozoa motility in all experimental groups with the addition of NP dissolved in 0.1% DMSO and 0.1% ETOH (P < 0.001 and P < 0.05). The lowest spermatozoa motility was found at doses > 100 μg/mL of NP in comparison with the control group. The viability of bovine spermatozoa detected by the MTT cytotoxicity assay was decreased significantly (P < 0.001) in all experimental groups with NP dissolved in 0.1% ETOH. The viability in groups with NP dissolved in 0.1% DMSO was significantly (P < 0.05) decreased at 1 μg/mL of NP and significantly decreased (P < 0.001) at doses > 10 μg/mL of NP. After 6 h of culture the MTT assay proved a negative effect of all NP doses the on cell viability. The obtained data clearly indicate the negative effect of NP as an endocrine disruptor on spermatozoa motility and viability, which should be seriously considered in the case of exposure to NP in animals and humans and as a reason of male reproductive dysfunction.

Effect of Semen Collection Methods on the Quality of Pre‐ and Post‐thawed Bali Cattle (Bos javanicus) Spermatozoa

This study was conducted to evaluate the response of Bali bulls (Bos javanicus) to different semen collection methods and their effects on fresh and post-thawed semen quality. The collection methods employed were electro-ejaculation (EE), transrectal massage (RM) and RM followed by EE (RM+EE). A total of 25 untrained Bali bulls (age between 2 and 4years old) were subjected to the different semen collection methods. Fresh semen samples from all the 25 bulls were evaluated for volume, pH, general motility, live/dead ratio and abnormality using the conventional method. For fresh and frozen samples collected by EE and RM from 10 bulls, computer-assisted semen analysis system was used for precise quantitative measurement of motility, velocity and forward progression. Accucell photometer was used to measure sperm concentration in all samples, regardless fresh and frozen. Semen samples were obtained 100% of the attempts using EE, 84% using RM and 96% using RM+EE. There were no differences among the collection methods for fresh semen quality characteristics, including motility, morphology and viability, but pH and volume were higher for EE than RM and RM+EE. Higher sperm concentration was observed in semen collected by RM than the other two methods. Different age groups (2-3 and >3-4years old) of the bulls did not show significant differences in volume, pH, sperm concentration, percentages in motility, live/dead ratio and normal sperm morphology. The quality of semen for general and progressive motility, VAP, VSL and VCL and acrosomal integrity after thawing was higher for RM than EE. In conclusion, Bali bulls appeared to respond best to EE and the combination of RM+EE than RM, as a method of semen collection, with a shorter time of stimulation required. Differences in age of the Bali bulls did not affect the semen quality.

Association of prostate cancer susceptibility variant (MSMB) rs10993994 with risk of spermatogenic failure

β-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. It has been identified that MSMB increased significantly in oligoasthenoteratozoospermic patients compared with fertile controls. We hypothesized that the functional polymorphism (rs10993994) of MSMB gene could be a risk factor for spermatogenic failure. For this study, 338 patients with idiopathic oligozoospermia or azoospermia and 382 fertile controls were recruited from an infertility clinic. Semen analysis was performed by computer-assisted semen analysis system. The functional polymorphism of MSMB gene was genotyped using TaqMan method. Sixty three seminal plasma samples were used to test the expression of MSMB by enzyme-linked immunosorbent assay (ELISA). The TT genotype and T allele were associated with an increased risk of idiopathic infertility with azoospermia (TT genotype: OR, 1.75; 95% CI, 1.03–2.95; T allele: OR, 1.34; 95% CI, 1.03–1.75). However, no differences were found in risk for the TT genotype or T allele among men with oligozoospermia. In addition, idiopathic infertile males have significantly higher MSMB expression levels than fertile controls. We present the first epidemiologic evidence supporting the involvement of common genetic polymorphism in MSMB gene in spermatogenic failure. These results suggest that men carrying the variant have an increased risk of spermatogenic failure associated with male infertility. Further studies are needed to confirm the roles of the polymorphism in idiopathic azoospermia and investigate the biological mechanism of elevated MSMB expression in infertile males.

Multivariate Analyses for Determining the Association of Field Porcine Fertility With Sperm Motion Traits Analysed by Computer‐Assisted Semen Analysis and With Sperm Morphology

This study investigates the association of semen traits with boar fertility. The fertility outcome (farrowing rate - FR and total piglets born - TB) of 14 boars was obtained from a field trial conducted during 10week of breeding period on a commercial farm using multiparous sows (n=948) through single-sire mating with 2×10(9) motile sperm cells per artificial insemination (AI) dose. Sperm motion parameters, evaluated with computer-assisted semen analysis system in raw and stored semen at 17°C for 240h, in addition to morphological sperm defects, measured on the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate cluster, canonical and discriminant analyses. Four clusters of boars were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The other boars were separated in two distinct clusters (four and seven boars), including boars with intermediate TB (12.64 and 13.22) but divergent values for FR (95.9% vs 91.8%). Semen traits with higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement and cytoplasmatic droplets. Through multivariate discriminant analysis, more than 80% of the 140 ejaculates were correctly classified into their own group, showing that this analysis may be an efficient statistical tool to improve the discrimination of potential fertility of boars. Nevertheless, the validation of the relationship between fertility and semen traits using this statistical approach needs to be performed on a larger number of farms and with a greater number of boars.

A computer system for a human semen quality assessment

An automatic computer system to assist assessment of a human semen quality is described. The hardware of the system consists of PC with frame grabber and CCD camera connected to an optical microscope with bright field optic. The software of the system consists of four modules: (1) acquisition of real time sequences of semen, (2) assessment of mobility o sperm cells, (3) calculation of density, and (4) morphology examination of sperm cells.There are few commercial CASA (Computer Assisted Semen Analysis) systems that measure a quantity of semen. Some of them measure only one factor of semen whereas others recognize sperm cells based on user-defined parameters. The described system calculates all factors required for semen quality assessment and recognizes sperm cells by its shape, structure and size.

Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders

The aim of this study was to assess the effect on boar spermatozoa quality of in vitro storage in short and long-term extenders by fluorescent multiple staining (FMS) and computer assisted semen analyzer (CASA). Fresh ejaculates from three healthy, sexually mature boars were diluted with equal volumes of six short-term or three long-term commercial extenders and stored at 19°C for 6 days (short-term) or 12 days (long-term). The integrity of spermatozoa membranes was analyzed by FMS using propidium iodide, 5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and fluorescein isothiocyanate-conjugated peanut agglutinin (PNA). The results obtained from this staining were compared with spermatozoa motility assessed by CASA. Our study showed that the number of viable spermatozoa with non-reacted acrosomes and intact mitochondria was positively correlated with the rate of motile spermatozoa (r2&gt;0.9) irrespective of the extender used. In all extenders the number of motile spermatozoa significantly decreased as preservation period increased (P&lt;0.05). FMS test is a potent indicator of sperm motility because it analyses mitochondrial integrity independently from observable alterations in motility. The best performing extenders were BTS for short-term storage and TRI-x-Cell for long-term storage.

Effects of Cooling Time on Membrane Integrity and Motility of Frozen‐Thawed Canine Spermatozoa using Two Different Commercial Egg Yolk–based Extenders at Two Different Cooldown Equilibration Times

Two commercially available egg yolk-based semen extenders, one marketed for human semen freezing (HEYE) and one marketed for canine semen freezing (CEYE), were used to cryopreserve semen from single ejaculates of 11 different dogs. For each extender, a 30- and a 60-min cooldown period was used prior to the addition of the extender containing glycerol and then immediately frozen in liquid nitrogen vapours. Sperm motility was measured using a computer-assisted semen analysis (CASA) system. Sperm intact membranes were measured using SYBER-14 and propidium iodide. Semen in the HEYE cooled for 60 min had a significantly greater percentage of intact membranes than the semen in the HEYE cooled for 30 min (p = 0.02). Semen in the HEYE cooled for 60 min had significantly greater total motility (p = 0.007) and progressive motility (p = 0.004) than semen cooled for 60 min in the CEYE and semen cooled for 30 min in the HEYE (total motility p = 0.02 and progressive motility p = 0.02). Semen cooled for 60 min in the CEYE did not differ significantly in total (p = 0.6) or progressive motility (p = 0.4) than semen cooled for 30 min in the CEYE. There was no difference in total (p = 0.8) or progressive motility (p = 0.8) between the semen cooled for 30 min in the HEYE and the semen cooled for 30 min in the CEYE.

Genetic variants in epoxide hydrolases modify the risk of oligozoospermia and asthenospermia in Han-Chinese population

ObjectivesEpoxide hydrolases are involved in detoxifying and excreting the environmental chemicals, which are associated with decreased semen quality and male infertility. We hypothesized that polymorphisms in epoxide hydrolases may be associated with risk of oligozoospermia and asthenospermia. Design and methodsIn this study, 468 fertile controls and 672 idiopathic male infertile patients were recruited. SNPstream and TaqMan assay were used to genotype four single nucleotide polymorphisms in EPHX1 and EPHX2. The semen analysis was performed by computer-assisted semen analysis system. ResultsOur results demonstrated that rs1042064 of EPHX2 was significantly associated with decreased risk of oligozoospermia (OR=0.65, 95% CI: 0.44–0.98) and asthenospermia (OR=0.66, 95% CI: 0.46–0.94). ConclusionsOur results provided evidence that genetic variants in epoxide hydrolases may modify the risk of oligozoospermia and asthenospermia in Han-Chinese population.

Brief Communication: Assessment and Validation of Nonspermicidal Condoms as Specimen Collection Sheaths for Semen Analysis and Assisted Conception

The choice of nonspermicidal sheaths for specimen collection for diagnosis and/or fertility treatment is limited. Those that are available tend to be relatively expensive and as a result, offered selectively to patients. This study describes the evaluation of three over-the-counter nonspermicidal condoms as alternatives to specialized specimen collection sheaths. Sheaths were incubated with motile sperm for up to 2 h and the percent Grade A motility, percent progressive motility and progressive velocity assessed using an ‘in-house’ computer-assisted semen analysis system every 30 min. Two of the sheaths tested, Pasante Naturelle (PN) and the Durex Avanti Ultima were shown to be highly toxic to sperm, leading to immobilization of most specimens within 1 h. However the loss of sperm motility when sperm were incubated with the Pasante Unique (PU) condom was no greater than with either the control (rigid 60 ml specimen container) or the Male Factor Pak (MFP), a commercially available specialized semen collection sheath. In conclusion, the PU brand of condom could be used as a cheaper alternative to the specimen collection sheaths currently available. Furthermore, some brands of condom sold as nonspermicidal are in fact toxic to sperm and will immobilize most specimens within 1 h.

Dose- and time-dependent effect of copper ions on the viability of bull spermatozoa in different media

The purpose of this in vitro study was to determine the effect of copper (Cu) on the motility and viability of spermatozoa in the presence of different culture media. Specifically, we examined the dose- and time-dependent effect of copper ions (Cu2+) on the motility and viability of spermatozoa during different time periods (Time 0 h, 1 h, 24 h). The percentage of motile spermatozoa and progressive motile spermatozoa was determined after exposure to concentrations of 3.9; 7.8; 15.6; 31.2; 62.5; 125; 250; 500; 1000 μM/L of Cu2+ using the Sperm VisionTM CASA (Computer Assisted Semen Analyzer) system. The cell viability was measured by the MTT (metabolic activity) assay. The initial spermatozoa motility in the presence of Cu2+ in physiological saline solution (PS) showed slight increased values at doses <31.20 μM/L of Cu2+ compared to the control group. The long-term cultivation (Time 24 h) reduced the average motility values in all experimental groups (P < 0.001) in comparison to the control group. Identical spermatozoa motility was detected for the percentage of progressive motile spermatozoa during all time periods. The culture medium containing 20 % bovine serum albumin (BSA), triladyl and 5 % glucose increased the overall percentage of spermatozoa motility after 1 h of cultivation. A concurrently maintained motility of spermatozoa at doses <62.50 μM/L of Cu2+ during the long-term in vitro cultivation confirms the protective effect of albumin. The cell viability was decreased significantly (P < 0.001) in all experimental groups with copper administration. The obtained data point out that Cu2+ at high doses is a toxic element on the spermatozoa motility, which subsequently disrupts the viability of cells. However, using a suitable culture medium containing an energy component- and protein-rich substrate, the spermatozoa motility could increase.

Studies on transdermal delivery of sinomenine hydrochloride through mouse skin treated by microneedle arrays

Objective To analyze the correlation of seminal plasma free testosterone and free L-carnitine with semen density,vitality,and motility,so as to investigate their effects on male fertility and their roles in the examination and treatment of male infertility.Methods Using chemiluminescence immunoassay method,LC-MS-MS and computer-assisted semen analysis system,we examined the seminal levels of free testosterone,free L-carnitine and sperm density,vitality,and motility in 192 infertile males(normozoospermia,n=36;oligozoospermia,n=48;asthenozoospermia,n=60;and oligoasthenozoospermia,n=48).The difference of free testosterone and free L-carnitine levels between different groups was analyzed,and the correlation of their levels with semen density,vitality,and motility was also analyzed using the SPSS 13.0 statistical software.Results The levels of free testosterone and free L-carnitine in the seminal plasma of the normozoospermia men were significantly higher than those of others(P0.05).Correlation analysis showed that there was positive correlation between seminal free testosterone with the sperm density(r=0.427,P0.05),seminal free L-carnitine with the sperm density(r=0.318,P0.01),and seminal free L-carnitine with sperm vitality,motility(r=0.641,P0.01;r=0.568,P0.01).Conclusion Seminal free testosterone level is correlated with the sperm density,and seminal free L-carnitine level is correlated with sperm density,motility and vitality;both levels can be taken as a biochemical index for the clinical diagnosis and treatment of male infertility.

Application of computer-assisted semen analysis to explain variations in pig fertility1

Sperm quality is often evaluated through computer-assisted semen analysis (CASA) and is an indicator of boar fertility. The aim of this research was to study the relationship between CASA motility parameters and fertility results in pigs. Insemination records and semen parameters from a total of 45,532 ejaculates collected over a 3-yr period were used. The statistical model for analysis of fertility data from these inseminations included factors related to sow productivity. The boar- and semen-related variance (direct boar effect) were corrected for the effects of individual boar, genetic line of the boar, age of the boar, days between ejaculations, number of sperm cells in an ejaculate, number of sperm cells in an insemination dose, and AI station. The remaining variance was analyzed if semen motility parameters had a significant effect. This analysis revealed significant (P < 0.05) effects of progressive motility, velocity curvilinear, and beat cross frequency on farrowing rate (FR). Total motility, velocity average path, velocity straight line, and amplitude of lateral head displacement affected (P < 0.05) total number of piglets born (TNB). Boar- and semen-related parameters explained 5.3% of the variation in FR and 5.9% of the variation in TNB. Motility parameters, measured by CASA, explained 9% of the boar- and semen-related variation in FR and 10% of the boar- and semen-related variation in TNB. Individual boar and genetic line of the boar affected (P < 0.0001) the variation in FR and TNB. No differences (P > 0.05) were observed between effects of AI stations on fertility outcome, underscoring the objectivity of the CASA system used. Motility parameters can be measured with CASA to assess sperm motility in an objective manner. On the basis of the motility pattern, CASA enables one to discriminate between the fertilizing capacity of ejaculates, although this depends on the genetic line of the boar used in AI stations.

Do CASA Systems Satisfy Consumers Demands? A Critical Analysis

Boar studs are often offered new technologies including several CASA (computer-assisted semen analysis) systems. However, independent information to assist their purchase decisions is not available. The systems accuracy and repeatability variation because of different factors can be evaluated through duplicate testing of semen samples and comparison of the results according to WHO standards for humans. This primary analysis and a thorough economic cost benefit evaluation will help to decide whether the purchase of a CASA system will be profitable for a boar stud. Our experience of implementing several CASA systems in the cooperative Dutch Artificial Insemination (AI) centres is used as a base for this discussion.

Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination

In order to obtain a more standardised semen motility evaluation, Varkens KI Nederland has introduced a computer assisted semen analysis (CASA) system in all their pig AI laboratories. The repeatability of CASA was enhanced by standardising for: 1) an optimal sample temperature (39 °C); 2) an optimal dilution factor; 3) optimal mixing of semen and dilution buffer by using mechanical mixing; 4) the slide chamber depth, and together with the previous points; 5) the optimal training of technicians working with the CASA system; and 6) the use of a standard operating procedure (SOP). Once laboratory technicians were trained in using this SOP, they achieved a coefficient of variation of < 5% which was superior to the variation found when the SOP was not strictly used. Microscopic semen motility assessments by eye were subjective and not comparable to the data obtained by standardised CASA. CASA results are preferable as accurate continuous motility dates are generated rather than discrimination motility percentage increments of 10% motility as with motility estimation by laboratory technicians. The higher variability of sperm motility found with CASA and the continuous motility values allow better analysis of the relationship between semen motility characteristics and fertilising capacity. The benefits of standardised CASA for AI is discussed both with respect to estimate the correct dilution factor of the ejaculate for the production of artificial insemination (AI) doses (critical for reducing the number of sperm per AI doses) and thus to get more reliable fertility data from these AI doses in return.

Standardization of computer-assisted semen analysis using an e-learning application

Computer-assisted semen analysis (CASA) is primarily used to obtain accurate and objective kinetic sperm measurements. Additionally, AI centers use computer-assessed sperm concentration in the sample as a basis for calculating the number of insemination doses available from a given ejaculate. The reliability of data is often limited and results can vary even when the same CASA systems with identical settings are used. The objective of the present study was to develop a computer-based training module for standardized measurements with a CASA system and to evaluate its training effect on the quality of the assessment of sperm motility and concentration. A digital versatile disc (DVD) has been produced showing the standardization of sample preparation and analysis with the CASA system SpermVision™ version 3.0 (Minitube, Verona, WI, USA) in words, pictures, and videos, as well as the most probable sources of error. Eight test persons educated in spermatology, but with different levels of experience with the CASA system, prepared and assessed 10 aliquots from one prediluted bull ejaculate using the same CASA system and laboratory equipment before and after electronic learning (e-learning). After using the e-learning application, the coefficient of variation was reduced on average for the sperm concentration from 26.1% to 11.3% (P ≤ 0.01), and for motility from 5.8% to 3.1% (P ≤ 0.05). For five test persons, the difference in the coefficient of variation before and after use of the e-learning application was significant (P ≤ 0.05). Individual deviations of means from the group mean before e-learning were reduced compared with individual deviations from the group mean after e-learning. According to a survey, the e-learning application was highly accepted by users. In conclusion, e-learning presents an effective, efficient, and accepted tool for improvement of the precision of CASA measurements. This study provides a model for the standardization of other laboratory procedures using e-learning.

Sperm detection in video frames of semen sample using morphology and effective ellipse detection method

CASA (Computer assisted semen analysis) systems are designed to assist Andrologist labour. Most available CASA systems are not accurate or so expensive. Therefore labours use manual methods to provide parameters. Although some companies have achieved appropriate accuracy, they have not released their methods. So proposing methods in this area might be useful for groups who intend to design new CASA system. One of the parameters which these systems compute is sperm count. In this paper we introduce our algorithm which can count sperms with an acceptable accuracy. Sperm count or concentration is one determinant parameter in male fertility. Our program preprocesses the video frame or image of semen sample under the microscope recorded by camera, then use morphology and effective ellipse detection method techniques to segment sperms and then count appropriate sperms.

Possibilities of using the European bison (Bison bonasus) epididymal spermatozoa collected post-mortem for cryopreservation and artificial insemination: a pilot study

BackgroundEuropean bison is the largest mammal in Europe with the population of approximately 4000 individuals. However, there is no report of post-mortem spermatozoa collection and cryopreservation from this species and the aim of this study was to test if the epididymal spermatozoa collected post-mortem from European bison are suitable for cryopreservation and artificial insemination (AI).MethodsEpididymides were collected post-mortem from two European bison bulls at age of 8 (bull 1) and 11 year (bull 2). Epididymal sperm was harvested by making multiple incisions in caudae epididymidis, which were then rinsed with extender. The left epididymis of bull 1 was rinsed with BioXcell (IMV, France), whereas the right epididymis of bull 1 and the right and left epididymides of bull 2 were rinsed with the extender based on Tris, citric acid, glucose, egg yolk, glycerol, antibiotics and distilled water (extender II). The diluted semen was cooled to 5 degrees C, and frozen in liquid nitrogen vapour. Then, properties of the frozen/thawed semen were examined with the use of computer-assisted semen analysis system, and thirty cows and nine heifers of domestic cattle were artificially inseminated.ResultsMotility of fresh spermatozoa collected from the right epididymis of bull 1 was 70% (spermatozoa diluted with extender II), and from the left one was 60% (spermatozoa diluted with BioXcell), whereas motility of fresh spermatozoa collected from bull 2 was 90% (spermatozoa diluted with extender II). Spermatozoa motility just after thawing were 11 and 13% in bull 1, respectively for spermatozoa collected from the left and right epididymis and 48% in bull 2. As a result of AI of domestic cows and heifers with the frozen/thawed European bison spermatozoa, two pregnancies were obtained in heifers. One pregnancy finished with a premature labour after 253 days of pregnancy, and the second one after 264 days of pregnancy.ConclusionsThis is the first report showing pregnancy in the domestic cattle following AI with frozen-thawed European bison spermatozoa collected post-mortem. The protocol of spermatozoa collection, dilution, and cryopreservation presented in this paper may be useful for the creating genetic resource bank in the European bison.

Variants of the EPPIN gene affect the risk of idiopathic male infertility in the Han-Chinese population

It has been identified that human epididymal protease inhibitor (EPPIN) plays a critical role in sperm function and male fertility. The aim of this study was to determine whether variants of the EPPIN gene are risk factors for idiopathic male infertility. All subjects, including 473 idiopathic infertile men and 198 fertile controls, underwent complete historical and physical examinations. Each subject donated 5 ml of peripheral blood for genomic DNA extraction and serum testosterone evaluation and an ejaculate for semen analysis. The semen analysis was performed by computer-assisted semen analysis system. The serum testosterone level was evaluated by radioimmunoassay. Four tagging single-nucleotide polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. We have demonstrated a significant decreased risk of idiopathic infertility with abnormal semen parameters in association with the variant rs2231829, and an increased risk of idiopathic infertility with abnormal semen parameters in association with the variant rs11594. However, among men with normal semen parameters, there were no differences in risk for these genotypes. Furthermore, no significant differences were found for the other variants, rs6124715 and rs2227290, on the risk of male infertility with normal or abnormal semen parameters. Similar serum testosterone levels among different EPPIN genotypes were observed for each group. These results suggest that different variants in the EPPIN gene may have different relationships with idiopathic male infertility and men carrying these variants have a decreased or increased risk of abnormal semen parameters associated with male infertility.

Profiles of sperm morphology and motility after discontinuous multiple-step Percoll density gradient centrifugation.

Morphology and motility are important parameters for assessing the fertilizing capacity of spermatozoa. This investigation reports a systematic study on the profiles of these parameters after percoll gradient centrifugation. Spermatozoa from normal human semen were fractionated by discontinuous Percoll gradients (30%, 45%, 75%, 90%). Spermatozoa washed with Earle's balanced salt solution were used as a control. After centrifugation, sperm morphology was evaluated according to strict criteria; motility was assessed by a computer-assisted semen analysis system. The results showed that the percentage of spermatozoa with normal morphology increased, while those with severe morphological defects decreased, as the density of Percoll increased. The percentages of motile spermatozoa and hyperactivated spermatozoa, and the velocity and amplitude of lateral head displacement of the spermatozoa were significantly higher in the 75% and 90% Percoll fractions than that in the 30% and 45% ones, and in the control. These results demonstrated that Percoll density gradient centrifugation enriched spermatozoa in terms of morphology and motility.