Asthenozoospermic Semen Samples Research Articles

Phosphatidylserine Levels in Seminal Plasma of Iraqi Infertile Men Using Direct and Indirect Swim Up Sperm Preparation Techniques

Phosphatidylserine (PS), exposed on viable sperm, is required for fertilization as it is recognized by specific receptors located on the microvilli of the oocyte to promote sperm: egg fusion. The heads of viable and motile sperm exhibit PS. With (PS) exposure progressively increasing during sperm transit through the epididymis. PS asymmetry on the leaflets of plasma membrane is arranged in a complex way, via multiple proteins functioning in opposite directions; further, PS is also essential for many intracellular trafficking events. The aim of this study was to evaluate the phosphatidylserine PS level in seminal plasma of infertile men before and after activation using Direct Swim Up (DSU) and Indirect Swim Up (IDSU) activation method and make a comparison between them. Forty infertile men were included in this study during their attendance to the infertility clinic at High Institute for Infertility Diagnosis and Assisted Reproductive Technologies, Al-Nahrain University. Normozoospermic or mild asthenozoospermic semen samples were used to choose the cases, according to WHO manual standards (1999 -2010). The collected semen samples obtained were divided into three parts: The first part prepared was as an in vitro sperm characterization before activation while the second and third parts were after activation using direct and indirect swim up techniques respectively. The level of seminal plasma phosphatidylserine was increased after the activation techniques from the baseline level before activation. Comparison of Seminal plasma phosphatidylserine levels between direct and indirect sperm activation technique were significantly higher in direct swim up activation technique than indirect one. Phosphatidylserine (PS) in semen plasma of infertile men was significantly increased after activation using Direct Swim Up DSU preparation method.

P-067 Characterization of seminal microbiota in patients with asthenozoospermic and normozoospermic using next-generation sequencing

Abstract Study question How are the semen microbiota compositions of patients with asthenozoospermic and normozoospermic different, According to data from 16S rRNA gene-specific Next-generation sequencing (NGS)? Summary answer Large numbers of microbes can be present in seminal fluid, and there are differences in the semen microbiota between asthenozoospermic and normozoospermic semen samples. What is known already Male factor is attributable in up to 50% of cases of infertility. Most studies on normal and abnormal sperm microbiota are based on 16S rRNA gene-specific next-generation sequencing (NGS). Microbiome analysis based on subunit 16S rRNA sequencing is a fast tool that can enable the identification of all the pathogenic microorganisms associated with semen in clinical pathology. Studies on the impact of semen micro-biomes in asthenozoospermic and normozoospermic samples could improve the results of assisted reproductive technologies. The major bacterial diversity in asthenozoospermic and normozoospermic samples belongs to the genera Lactobacillus, Prevotella, Staphylococcus, and Anaerococcus. Study design, size, duration Two eighty patients with their own semen samples were included in the study. The study population consists of patients attending to the “Pacific IVF Center” Pacific Medical University and Hospital (Udaipur, India) from November 2020 to October 2022. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 127) was asthenozoospermic, and Group 2 (n = 153) had normozoospermic semen samples, Patients were aged between (20-45 years). Ethical approval was obtained from the institute. Participants/materials, setting, methods Genomic DNA was extracted from samples using commercially available (Qiagen, DNeasy Power Soil kit). The amount of extracted DNA was measured using a Nanodrop spectrophotometer (Thermo Scientific). The microbiota of semen was analyzed using 16S ribosomal RNA (rRNA) gene amplification (MinION) Oxford Nanopore Ltd. Bioinformatics analysis was performed using QIIME2 and Microbiome Analyst packages. Alpha, beta diversity, and taxonomic characterization were compared for the seminal microbiome in asthenozoospermic and normozoospermic semen samples. Main results and the role of chance Different bacterial communities were detected when asthenozoospermic and normozoospermic semen samples were analyzed. In patients with asthenozoospermic parameters, a higher alpha diversity index tendency was found. In normozoospermic semen samples, a p-value of bacterial diversity was non-significant (p = 0.14 for the Shannon index), and significant differences in asthenozoospermic semen samples (p = 0.01 for the Shannon index). In the beta diversity analysis, no significant differences were observed between both groups Relative abundance analysis identified the bacterial communities, Lactobacillus, Streptococcus, and Ureaplasma in normozoospermic semen samples, and Anaerococcus and Gardnerella in asthenozoospermic samples, Comparison of asthenozoospermic and normozoospermic samples with alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Relative abundance in normozoospermic semen samples identified the bacterial communities, Lactobacillus (p = 3.70E-4), Prevotella (p = 8.11E-4), and in asthenozoospermic semen samples Anaerococcus (p = 0.004) and Gardnerella (p = 0.004). Limitations, reasons for caution The limited sample size is the primary constraint of this study. Larger studies with a larger sample size are needed to confirm the distinct microbiome patterns identified in asthenozoospermic and normozoospermic samples in connection to male infertility. Wider implications of the findings Further research could determine the detection rate of the described bacterial diversity in semen with other pathologies. Determining the relationship between sperm microbiota characteristics and infertility could enable the development of new algorithms for treating patients with reproductive disorders, An abnormal seminal microbiome appears to be strongly correlated with infertility. Trial registration number N/A

Development and integration of LensHooke® R10 for automatic and standardized diagnosis for sperm DNA fragmentation.

The sperm chromatin dispersion assay is commonly used to assess sperm DNA integrity. This approach is time-consuming, demonstrates poor chromatin preservation, and provides an ambiguous and unstandardized evaluation of fragmented chromatin. We aimed to (i) develop an optimized sperm chromatin dispersion assay with reduced operation time, (ii) validate R10 test accuracy by comparing it to a conventional sperm chromatin dispersion assay, and (iii) standardize the sperm DNA fragmentation analysis procedure by integrating artificial intelligence optical microscopic technology. This cross-section study included 620 semen samples. Aliquots were analyzed by a conventional Halosperm® G2 assay (G2) and LensHooke® R10 assay (R10). The DNA fragmentation index was scored manually, and R10 slides were automatically determined by a LensHooke® X12 PRO semen analysis system (X12). We demonstrated significant improvements in total assay time (40 vs. 72min, p<0.001) and in the halo-cytological resolution using R10 compared to G2. Comparing the G2 and R10, DNA fragmentation index results demonstrated good agreement between the two methods (Spearman's rank correlation, rho=0.8517, p<0.0001). We introduced the integration of an auto-calculation system to diagnose sperm DNA fragmentation. X12 interpretation showed excellent agreement with manual interpretation (Spearman's rank correlation, rho=0.9323, p<0.0001), but had a low coefficient of variation compared to manual interpretation (4% for R10 by X12 vs. 19% for R10 by manual scoring vs. 25% for G2 by manual scoring). DNA fragmentation index was more correlated with total motility (coefficients=-0.3607, p<0.0001) than sperm morphology and was positively associated with asthenozoospermic semen samples (p=0.0001). The R10 sperm chromatin dispersion assay combined with the X12 semen analysis system is faster, more objective, and provides standardization for sperm DNA fragmentation.

Presence of acetylated α-tubulin in human sperm nuclei: A contributor to sperm heterogeneity

The global rise in male infertility necessitates a constant search for underlying causes, diagnostics and corrective treatments. Sperm cells are irreplaceable cells for the process of reproduction. Intrauterine insemination (IUI) is a simple and non-invasive technique, most commonly used to overcome both unexplained and mild male-factor infertility. Even though spermatozoa may be classified as “normal” in terms of morphology, motility and concentration, they could be defective at the subcellular level. Protein lysine acetylation within human sperm, a major post-translational modification of tubulin, is suspected to be a cause of sperm abnormality but is not fully understood. An examination of α-tubulin and acetylated α-tubulin immunopresence, spatial distribution and co-localisation in normozoospermic and asthenozoospermic semen samples was conducted to determine if acetylated α-tubulin could be a biomarker of sperm heterogeneity to better predict sperm fertility potential for IUI outcome. Acetylated α-tubulin was present in both sperm subcompartments, tail and nucleus of normozoospermic samples. We found more immunopositivity for acetylated α-tubulin in the sperm tail and less in nucleus of asthenozoospermic compared to normozoospermic samples. Hence, the acetylated α-tubulin in sperm may contribute to sperm nuclei heterogeneity and serve as a novel molecular biomarker.

Sperm motility in asthenozoospermic semen samples can be improved by incubation in a continuous single culture medium (CSCM®)

ABSTRACT Standard protocols for clinical in vitro fertilization (IVF) laboratories recommend incubating semen at 37°C in 5% CO2 without strictly specifying which medium should be used or for how long. This study aimed to test the most common different incubation media used in Latin American andrology and micromanipulation laboratories and verify which, if any, is the most appropriate medium to improve asthenozoospermic semen samples’ motility in the infertile male population. Ejaculates (136) collected from asthenozoospermic men were divided into two cohorts with similar characteristics (cohort 1; n = 28 and cohort 2; n = 108). Cohort 1 was used to evaluate the optimal incubation time with regard to unprepared asthenozoospermic sample sperm motility. After defining an optimal incubation period of 2 h, cohort 2 was used to evaluate which of the four media commonly used in IVF clinics (continuous single culture medium = CSCM®; SpermRinse medium = SR®; in vitro fertilization medium = G-IVF® and human tubal fluid medium = HTF®) was preferred for semen samples from asthenozoospermic patients. Overall, it was determined that a 2-h incubation in CSCM® medium led to the highest asthenozoospermic sperm motility. Thus, this simple, cost-effective, easily reproducible protocol could prove extremely useful for andrology laboratories working with IVF clinics dealing with asthenozoospermic semen specimens. This is particularly relevant since the incidence of the latter is on the rise as semen quality decreases around the globe. Abbreviations: ANOVA: Analysis of variance; ARTs: Assisted reproductive techniques; BWW: Biggers, Whitten, and Whittingham; CO2: Carbon dioxide; CPM: counted per minute; CSCM: Continuous Single Culture Medium; DAB: 3.3′- diaminobenzidine; DFI: DNA Fragmentation Index; DMSO: Dimethyl sulfoxide; G-IVF: In Vitro Fertilization Medium; GSH: Glutathione; GPx: glutathione peroxidase; HDS: High DNA Stainability; HSA: Human Serum Albumin; HTF: Human Tubal Fluid; HYP: Hyperactivity; ICSI: Intracytoplasmic sperm injection; IUI: Intrauterine insemination; IVF: in vitro fertilization; LIN: Linearity; ROS: Reactive Oxygen Species-level; SC: Sperm concentration; SCA: Sperm Computer Analysis; SCSA: Sperm Chromatin Structural Assay; SR: SpermRinse medium; SSS: Synthetic Serum Substitute; STR: Straightness; SOD: superoxide dismutase; TNE: Tris-Borate-EDTA; TSC: Total sperm count; VAP: Mean velocity; VCL: Curvilinear velocity; VSL: Linear velocity; WHO: World Health Organization; WOB: Wobble; spz: spermatozoa; AO: antioxidant

Caffeine increased progressive motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples and enhanced activity of seminal creatine kinase.

Even though the effect of caffeine on humans' health has been revealed in various research studies, its effect on semen quality has yet to be well explained. Here, we measured the effect of caffeine at 1, 5, 10 and 20mM on motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples, level of seminal nitric oxide, chelation of seminal calcium ions and activity of seminal creatine kinase. Fifty-one normozoospermic and ten asthenozoospermic semen samples were recruited in this study. Sperm motility was evaluated by Makler counter, and seminal nitric oxide, seminal-free calcium and activity of seminal creatine kinase were measured spectrophotometrically. Caffeine at 10mM significantly (p<.05) increased progressive motility of spermatozoa in both tested groups. Also, caffeine significantly increased (p<.05) activity of creatine kinase and insignificantly (p>.05) altered nitric oxide and free calcium levels in seminal plasma. In conclusion, progressive motility of human spermatozoa was found to be higher in the presence of caffeine at 10mM in normozoospermic and asthenozoospermic semen samples; this increase, albeit partially, could be due to increased activity of seminal creatine kinase, but not to increased production of nitric oxide or chelation of free calcium ions.

Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients

As the largest proportion of male infertility population, asthenozoospermia patients often resort to sperm cryopreservation to preserve fertility as well as to enrich motile sperm for assisted reproductive techniques (ART), although it may cause some cryodamage during the freezing–thawing process. The objective of this study was to investigate whether mitochondrial antioxidant Mito-Tempo was effective in preventing cryodamage of asthenozoospermic spermatozoa. Asthenozoospermic semen samples were collected and cryopreserved in media supplemented with different concentrations (0.0, 1.0, 10 and 100 μM) of Mito-Tempo. We measured sperm motility, viability, membrane integrity, DNA fragmentation, mitochondrial membrane potential, oxidation product, and antioxidant enzymes activities. Supplementation of the cryopreservation media with Mito-Tempo (10 and 100 μM) induced a significant improvement in sperm viability, motility, membrane integrity, mitochondrial membrane potential and chromatin integrity (P < 0.05). Significant enhancement of antioxidant enzymes activities accompanied by the decreased formation of oxidation products (ROS and MDA) was also observed in groups supplemented with Mito-Tempo (10 and 100 μM). It is concluded that mitochondria targeted antioxidant Mito-Tempo alleviates cryodamage by regulating intracellular oxidative metabolism in spermatozoa from asthenozoospermic patients after cryopreservation.

Human sperm motility: a molecular study of mitochondrial DNA, mitochondrial transcription factor A gene and DNA fragmentation.

Alterations affecting the mitochondrial genome and chromatin integrity of spermatozoa impair male reproductive potential. This study aimed to evaluate the impact of mitochondrial DNA (mtDNA) copy number alterations on sperm motility and on the molecular mechanism regulating the number of mtDNA copies, through analysis of mitochondrial transcription factor A (TFAM) gene expression. It also investigated any correlation between mtDNA copy number and sperm DNA fragmentation (SDF). Sixty-three asthenozoospermic semen samples (Group A) and 63 normokinetic semen samples (Group N) were analysed according to WHO (WHO laboratory manual for the examination and processing of human semen, World Health Organization, Geneva, 2010). Sperm mtDNA copy number and TFAM gene expression were quantified by real time quantitative polymerase chain reaction. SDF was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. The mtDNA copy number was higher in asthenozoospermic semen samples and was negatively correlated with sperm concentration, total sperm number and total motile spermatozoa. The caseload showed a global negative correlation of TFAM gene expression with total motile sperm and a positive correlation with abnormal forms, SDF and mtDNA copy number, but this was not confirmed within each subgroup. SDF was significantly increased in asthenozoospermic samples and correlated with abnormal forms. No correlation was found between SDF and mtDNA copy number. Our results suggest a potential role of mtDNA content as an indicator of semen quality and support the hypothesis that dysregulation of TFAM expression is accompanied by a qualitative impairment of spermatogenesis. Since mtDNA copy number alterations and impaired chromatin integrity could affect reproductive success, these aspects should be evaluated in relation to assisted reproductive techniques.

Molecular study of human sperm RNA: Ropporin and CABYR in asthenozoospermia.

Sperm motility is an essential aspect of human fertility. Sperm contain an abundance of transcripts, thought to be remnants of mRNA, which comprise a genetic fingerprint and can be considered a historic record of gene expression during spermatogenesis. The aberrant expression of numerous genes has been found to contribute to impaired sperm motility; these include ROPN1 (rhophilin associated tail protein 1), which encodes a component of the fibrous sheath of the mammalian sperm flagella, and CABYR (calcium-binding tyrosine-(Y)-phosphorylation-regulated protein), which plays an important role in calcium activation and modulation. The aim of this study was to investigate ROPN1 and CABYR gene co-expression in asthenozoospermic semen samples in comparison with normozoospermic samples. We studied 120 semen samples (60 normozoospermic and 60 asthenozoospermic) from Caucasian patients attending our centre for an andrological check-up. Total RNA was extracted from purified spermatozoa with RNeasy mini kit. ROPN1 and CABYR mRNA expression was analysed using RT-qPCR. Continuous variables were described as means±standard deviations. ROPN1 and CABYR mRNA were simultaneously downregulated in asthenozoospermic in comparison with normozoospermic samples. There was also a positive correlation between total progressive motility and ROPN1 and CABYR gene expression and between total motile sperm number and ROPN1 and CABYR gene expression. The results demonstrated downregulation of both ROPN1 and CABYR in asthenozoospermic samples and importantly, a positive correlation between the expression of the two genes, suggesting that ROPN1 and CABYR co-expression is a prerequisite for normal flagellar function and sperm motility.

In-Vitro Application of Pentoxifylline Preserved Ultrastructure of Spermatozoa After Vitrification in Asthenozoospermic Patients.

To evaluate the effect of in vitro application of pentoxifylline (PX) on sperm parameters and ultrastructure after vitrification in asthenozoospermic patients. A total of 30 asthenozoospermic semen samples (aged 25-45 years) were divided into four groups before vitrification, after vitrification, control (without PX) and experimental (with PX). In experimental group, each sample was exposed for 30 min to 3.6mmol/l PX and the control group without any treatment apposing in 370C for 30 min. After incubation, the samples were washed and analyzed again. Vitrification was done according to straw method. Eosin-nigrosin and Papanicolaou staining were applied for assessment of sperm viability and morphology, respectively. The samples without PX and post treatment with PX were assessed by transmission electron microscopy (TEM). A significant decrease in sperm motility (P &le; .001), morphology (11.47 &plusmn; 2.9 versus 6.73 &plusmn; 2.01) and viability (73.37 &plusmn; 6.26 versus 54.67 &plusmn; 6.73) was observed post vitrification, but sperm motility (19.85 &plusmn; 4.75 versus 32.07 &plusmn; 5.58, P &le; .001) was increased significantly following application of PX. This drug had no significant (P &gt;.05) detrimental neither negative effect on ultrastructure acrosome, plasma membrane and coiled tail statues of spermatozoa. Vitrification had detrimental effects on sperm parameters, but PX reversed detrimental effects on sperm motility. However, PX had no alteration on ultrastructure morphology of human spermatozoa after vitrification.

Effects of in-vitro application of pentoxifylline on the morphology of human spermatozoa after vitrification in asthenozoospermic patients

Cryopreservation of human spermatozoa is widely used in many assisted reproduction units to preserve male fertility [1]. Vitrification is based on the ultrarapid freezing and is routinely assayed for cryopreservation in assisted reproductive technology. Mohamed [2] showed that cryopreservation significantly affects progressive motility, viability and mitochondrial membrane potential of spermatozoa. Pentoxifylline (PX) is a phosphodiesterase considered to be a sperm movement enhancer, hyperactivation agent, inhibitor of reactive oxygen species and acrosome reaction-improving agent. The aim of our study was to evaluate the effect of in-vitro application of PX on sperm parameters and ultrastructure after vitrification. A total of 30 asthenozoospermic semen samples were selected and divided into two groups after vitrification: control (without PX) and experimental (with PX). A significant decrease in sperm motility, morphology and viability was observed post vitrification, but sperm motility was increased significantly following application of PX. On the other hand, PX did not exert any significant effect on the ultrastructure of the acrosome, plasma membrane and tail of vitrified spermatozoa.

Effect of nerve growth factor on sperm quality in asthenozoosprmic men during cryopreservation.

BackgroundAlthough routinely used in assisted reproductive technology, human sperm cryopreservation is not an entirely successful procedure. This study determined the effect of nerve growth factor (NGF) supplementation of cryopreservation medium on post-thaw viability, motility, intracellular nitric oxide (NO) concentration, and DNA fragmentation of human spermatozoa in asthenozoospermic men.MethodsSemen samples were collected from 25 asthenozoosprmic men and divided into the following groups (n = 5/group): fresh semen (control); frozen-thawed semen without treatment; frozen-thawed semen with NGF treatment (0.5, 1, and 5 ng/ml). Prior to dividing the asthenozoospermic samples, 200 μl of each sample was collected for NGF content assessment by ELISA and then compared with normozoospermic semen samples (25 normozoospermic men). Sperm motility and viability were assessed according to WHO criteria. Furthermore, intracellular nitric oxide and DNA fragmentation were evaluated by Flow Cytometry.ResultsNGF content was significantly higher in normozoospermic compared with asthenozoospermic men. Cryopreservation of asthenozoospermic semen samples significantly decreased sperm viability and motility, and increased intracellular nitric oxide concentration and DNA damage (p < 0.01). In asthenozoospermic frozen–thawed samples treated with 0.5 ng/ml exogenous NGF, we observed a significantly increased viability, motility, and decreased DNA fragmentation (p < 0.05), but intracellular nitric oxide concentration was not reduced. The other high doses (1 and 5 ng/ml) had no significant effect on the variables.ConclusionSupplementation with exogenous NGF could have partial and limited protective effect during cryopreservation of human spermatozoa but further research is needed to evaluate the possible clinical applications.

TAT-peroxiredoxin 2 Fusion Protein Supplementation Improves Sperm Motility and DNA Integrity in Sperm Samples from Asthenozoospermic Men

We compared levels of peroxiredoxin 2 in semen samples from normozoospermic and asthenozoospermic men. The potential effects of TAT-peroxiredoxin 2 fusion protein on sperm motility and DNA integrity were also evaluated. Semen samples were obtained from 50 normozoospermic and 50 asthenozoospermic men. Lipid peroxidation of semen was determined using a commercial malondialdehyde kit. Sperm DNA fragmentation was evaluated by TUNEL assay. Western blot and immunofluorescence were performed to detect the amount of peroxiredoxin 2 protein in seminal plasma and spermatozoa. Sperm motility, DNA damage and levels of reactive oxygen species were evaluated after TAT-peroxiredoxin 2 fusion protein supplementation to the sperm suspension for 2 and 12 hours of incubation. In asthenozoospermic semen samples a significantly higher level of malondialdehyde and DNA damage was discovered. However, the expression of peroxiredoxin 2 was significantly lower in seminal plasma and spermatozoa compared with that of normozoospermic men. TAT-peroxiredoxin 2 fusion protein was successfully prepared and delivered to the spermatozoa. Interestingly adding TAT-peroxiredoxin 2 in asthenozoospermic sperm suspension effectively defended against the decrease in progressive motility and the increase in DNA damage. This study shows that supplementation of TAT-peroxiredoxin 2 fusion protein in the sperm suspension from asthenozoospermic men effectively improved sperm motility and DNA integrity by reducing levels of reactive oxygen species. Therefore, we speculate that peroxiredoxin 2 may have an important role as an antioxidant defense in semen and would provide new prevention and therapy alternatives for asthenozoospermia.

A proteomic analysis on human sperm tail: comparison between normozoospermia and asthenozoospermia.

Asthenozoospermia is a common cause of human male infertility characterized by reduced sperm motility. The molecular mechanism that impairs sperm motility is not fully understood. This study proposed to identify novel biomarkers by focusing on sperm tail proteomic analysis of asthenozoospermic patients. Sperm were isolated from normozoospermic and asthenozoospermic semen samples. Tail fractions were obtained by sonication followed by Percoll gradient. The proteins were extracted by solubilization and subjected to two-dimensional gel electrophoresis (2-DE); then, the spots were analyzed using Image Master 2D Platinum software. The significantly increased/decreased amounts of proteins in the two groups were exploited by matrix-assisted laser desorption-ionization time-of-flight/time-of-flight (MALDI-TOF-TOF) mass spectrometry. Three hundred ninety protein spots were detected in both groups. Twenty-one protein spots that had significantly altered amounts (p < 0.05) were excised and exploited using MALDI-TOF-TOF mass spectrometry. They led to the identification of the following 14 unique proteins: Tubulin beta 2B; glutathione S-transferase Mu 3; keratin, type II cytoskeletal 1; outer dense fiber protein 2; voltage-dependent anion-selective channel protein 2; A-kinase anchor protein 4; cytochrome c oxidase subunit 6B; sperm protein associated with the nucleus on the X chromosome B; phospholipid hydroperoxide glutathione peroxidase-mitochondrial; isoaspartyl peptidase/L-asparaginase; heat shock-related 70kDa protein 2; stress-70 protein, mitochondrial; glyceraldehyde-3-phosphate dehydrogenase, testis-specific and clusterin. Fourteen proteins present in different amounts in asthenozoospermic sperm tail samples were identified, four of which are reported here for the first time. These proteins might be used as markers for the better diagnosis of sperm dysfunctions, targets for male contraceptive development, and to predict embryo quality.

Vitamin E supplementation in semen-freezing medium improves the motility and protects sperm from freeze-thaw–induced DNA damage

This study evaluated the effect of vitamin E supplementation to cryoprotective media on post-thaw motility and DNA integrity of normozoospermic and asthenozoospermic semen samples. The results of this study indicate that supplementation of vitamin E (5 mM) significantly improves the post-thaw motility and DNA integrity in normozoospermic and asthenozoospermic semen samples.

Processing of semen can result in increased sperm DNA fragmentation

Processing of semen for assisted reproductive technologies (ART) entails a number of procedures that include semen liquefaction, removal of seminal plasma by centrifugation, incubation, and cryopreservation. The results of this study indicate that incubation of semen at room temperature and semen cryopreservation can result in increased levels of sperm DNA fragmentation.

Post-coital test in subfertile men.

Twenty-two patients with border-line spermiograms (moderate oligoasthenozoospermia or asthenozoospermia) were studied by the post-coital test (PCT) to determinate the behaviour in vivo of semen with modest dysspermia (spermatozoa counts between 10 and 20 million/ml, with 20-40% forward motility). All the female partners were normal morphologically and functionally. The PCT were always carried out when the cervical mucus was favorable (mean score 12.2-12.3) and the endocervical pH was 7.25-7.30. The positive results for the asthenozoospermic semen samples were 6.75% and for the oligozoospermic one 10%, when the classification were based on the W.H.O. criteria. When the semen samples were classified according to Kremer's criteria positive results were found in 37.5% and 40% of the cases respectively. The effects of both cervical mucus properties and pH on the results of the PCT were found to be important. The high percentage of the favorable results emphasizes the usefulness of completing studies of border-line spermiograms with an in vivo test, since it is difficult to define normality and predict the fertility potential from semen examination alone.

Identification of proteomic differences in asthenozoospermic sperm samples

Asthenozoospermia is one of the most common findings present in infertile males, but its aetiology remains unknown in most cases. Present proteomic tools now offer the opportunity to identify proteins which are differentially expressed in asthenozoospermic semen samples and potentially involved in infertility. We compared the expression of 101 sperm protein spots in 20 asthenozoospermic samples to that of 10 semen donor controls using two-dimensional proteomic analysis. Seventeen protein spots have been identified at different amounts in the asthenozoospermic samples compared with controls. These are cytoskeletal actin-B, annexin-A5, cytochrome C oxidase-6B, histone H2A, prolactin-inducible protein and precursor, calcium binding protein-S100A9 (2 spots), clusterin precursor, dihydrolipoamide dehydrogenase precursor, fumarate hydratase precursor, heat shock protein-HSPA2, inositol-1 monophosphatase, 3-mercapto-pyruvate sulfurtransferase/dienoyl-CoA isomerase precursor, proteasome subunit-PSMB3, semenogelin 1 precursor and testis expressed sequence 12. The detected amount of these proteins enabled the grouping of asthenozoospermic sperm samples in an unsupervised clustering analysis. We have identified several proteins present at different amount in asthenozoospermic sperm samples. These proteins could be candidates towards the development of diagnostic markers, and open up the opportunity to gain further insight into the pathogenic mechanisms involved in asthenozoospermia.

Antioxidant supplementation in vitro does not improve human sperm motility

Objective: To determine the effects of supplementation of preparation media with ascorbate and α-tocopherol on subsequent sperm motility and reactive oxygen species production. Design: Prospective study to analyze postpreparation human sperm motility parameters and reactive oxygen species production following antioxidant supplementation. Setting: Andrology Laboratory, Royal Maternity Hospital, Belfast, Northern Ireland. Patient(s): Sixty patients attending the Andrology Laboratory for semen analysis. Intervention(s): Normozoospermic and asthenozoospermic semen samples (n = 10 for each control and antioxidant group) were prepared by Percoll density centrifugation (95%–47.5%) in media supplemented with ascorbate or α-tocopherol to different concentrations within physiologic levels. Controls were included that were not exposed to antioxidant. Main Outcome Measure(s): Sperm motility parameters were assessed using computer-assisted semen analysis. The generation of reactive oxygen species was determined using luminol-dependent chemiluminescence. Result(s): The production of reactive oxygen species by sperm was reduced by supplementation in vitro with ascorbate and α-tocopherol. However, progressive motility, average path velocity, curvilinear velocity, straight-line velocity, and linearity were decreased significantly, with the greatest inhibition observed with the highest concentrations of antioxidants. Conclusion(s): Supplementation of preparation media with ascorbate and α-tocopherol, either singly or in combination, is not beneficial to sperm motility.

A new method of the electrolyte-free long-term preservation of human sperm at 4°C

To develop a new method for the long-term preservation of human sperm. Andrology laboratory of our hospital. Thirty-one normal and 19 asthenozoospermic semen samples obtained from patients attending our infertility clinic. The average sperm motility was 70.2% and 36.0% in the normal and asthenozoospermic groups, respectively. Ejaculated sperm were centrifuged and washed using the electrolyte-free Percoll gradient and then were preserved at 4 degrees C. The motility of the preserved sperm was analyzed using computer-assisted semen analyzer after the addition of Ham's modified F-10 (GIBCO, Grand Island, NY). In the normal group, motility rate after the addition of Ham's F-10 for 1, 2, and 4 weeks of preservation was 65.4%, 40.4%, and 5.5%, respectively. In the asthenozoospermic group, motility rate after 1 and 2 weeks of preservation was 31.3% and 18.1%, respectively. Preservation solutions containing sodium or potassium decreased motility after preservation. Restoration of preserved sperm was not achieved by incubation alone; however, reinitiation was induced by incubation together with Ham's F-10. Human sperm in the electrolyte-free solution survived for a long period of time at 4 degrees C and reinitiation of sperm motility after preservation required the addition of Ham's F-10.