Biomarker Of Sperm Quality Research Articles

Telomeric RNAs, TERRA, as a Potential Biomarker for Spermatozoa Quality.

Characterization of long non-coding telomeric repeat-containing RNAs in sperm of normozoospermic and oligoasthenozoospermic men as new biomarker of idiopathic male infertility. We conducted an observational prospective study with two groups of men with normal or orligoasthenozoospermic spermiogram, aged 40 and above. Fertility parameters were analyzed in men undergoing intracytoplasmic sperm injection with donor oocytes, to avoid the female factor. Telomeric RNAs and telomere length were measured by quantitative fluorescent in situ hybridization. Data from seminal parameters and in-vitro fertilization were assessed according to IVIRMA protocols. Patients with oligoasthenozoospermia, who had worse seminal parameters, also obtained embryos with lower inner-cell-mass quality (p = 0.04), despite using donor oocytes. While mean levels of telomeric RNAs were similar for both groups, the percentage of spermatozoa with more than 3 foci was higher in oligoasthenozoospermic men (p = 0.02). Regarding telomere length, oligoasthenozoospermic men had shorter mean, a higher accumulation of short telomeres (15th percentile; p = 0.03) and a lower percentage of very-long telomeres (85th percentile; p = 0.01). Finally, a positive correlation was found between telomeric-RNAs intensity and total progressive motility in the spermatozoa of normozoospermic patients (r = 0.5; p = 0.03). Telomeric parameters were altered in the spermatozoa of the oligoasthenozoospermic group, which also showed lower quality embryos. Interestingly, in the normozoospermic group, a correlation was found between progressive motility and telomeric RNA levels, suggesting that they could be a good biomarker of sperm quality. Further studies are required to confirm these results and translate them into the clinical practice.Trial registration number: 1711-MAD-109-CB, 07/07/2021.

P-029 Genome-wide microRNA expression profiling in human spermatozoa and its relation to sperm quality

Abstract Study question Does the microRNA (miRNA) expression profile in human spermatozoa change with their morphological appearance? Summary answer This study revealed miRNA expression associated with good-quality sperm classified based on their morphology. These miRNAs could represent efficient biomarkers to evaluate male fertility status. What is known already We previously published a scoring scale where the spermatozoa with a Score 6 (Good spermatozoon) display normal head shape with symmetrical nuclear no extrusion and/or no invagination of the nuclear membrane, without any vacuole and normal base. The spermatozoa with a Score 0, display a nuclear-shape disorder with an abnormal base and at least one large vacuole (Bad spermatozoon). Despite the great magnitude of studies investigating the miRNA role in spermatogenesis, limited work has been done to evaluate it as a potential biomarker for sperm quality in relation to their morphology. Study design, size, duration In a prospective study from March 2023 to May 2023, twenty semen samples were collected. The samples were categorized according to their scoring morphology into Score 6 and Score 0. Ten samples (five with a Score 6 and five with a Score 0) were used for the analysis by microarray and ten (five with a Score 6, and five with a Score 0) for the miRNA expression analysis by RT-qPCR. Participants/materials, setting, methods Semen samples were scored according to their high-magnification morphology and stored at -80 °C. We used miRNA microarray chip to analyze miRNA profile. Statistical significance was evaluated by Significance Analysis of Microarrays, screened by Fold change>2 and p-values ≤ 0.05. The correlation between miRNAs and their corresponding mRNA targets was analyzed using in silico prediction algorithms. The methylome was studied using the DNA sequencing method. TaqMan miRNA assays were used to confirm the miRNA results. Main results and the role of chance We perform a robust transcriptomic analysis to define unique miRNA signatures able to identify the spermatozoa that have high scores regardless of morphological appearance at high magnification. There were 86 significantly overexpressed in Score 6 and 21 miRNAs in Score 0. Score 6-spermatozoa had significantly higher levels of hsa-miR-132 (FCx41, p=1.68E-8), hsa-miR-1973 (FCx32, p=6.19E-8), hsa-miR-34 (FCx30, p=8.43E-8), hsa-miR-25 (FCx27, p=1.30E-6), hsa-miR-342 (FCx24, p=1.57E-5), hsa-miR-15 (FCx14, p=8.56E-6), hsa-miR-125 (FCx13, p=6E-4), hsa-miR-30 (FCx12, p = 3.75E-6), than Score 0. The miRNAs overexpressed in Score 6 are predicted to target 910 messenger RNA involved in biological functions of the spermatozoa and several signaling pathways, such as mTOR, p53, and MAPK signaling pathways, as well as cell Intrinsic pathway for apoptosis, cellular senescence, and oxidative stress. Whereas genes targeted by score 0-miRNAs were implicated in the regulation of cellular growth and proliferation. Our data indicate a difference in methylation of the promoter region of miRNAs, and their validated target genes were observed between score 6 and score 0. Aberrant expression of the Score 6-miRs or their targets could be due to the methylation status of the promoter region and indicative of a defect in spermatogenesis. Limitations, reasons for caution This work provides a foundation for the interpretation of miRNA changes associated with spermatozoa morphological appearance. The data must be validated by TaqMan miRNA assays in a higher number of semen samples. Moreover, the molecular network of miRNA-targeted gene pathways also merits investigation. These experiments are currently ongoing. Wider implications of the findings The profiling of miRNAs repertoire in scored spermatozoa morphology opens new perspectives to explore male fertility status and provides a biomarker panel for sperm analysis during the ART procedure. Larger well-controlled investigations employing microRNAome approaches are needed to validate these conclusions. Trial registration number not applicable

Effect of probiotics and prebiotics on the composition of the equine fecal and seminal microbiomes and sperm quality: A pilot study

Probiotic and prebiotic effects on equine semen and gastrointestinal microbiome composition and sperm quality are unknown. This study aimed to evaluate the effects of pre-, pro- or synbiotic supplementation on fecal and semen microbiome composition and sperm quality parameters of stallions. This Latin square crossover trial involved four miniature pony stallions receiving control diet only, or addition of a pro-, pre- or synbiotic formulation. Full-length 16S rRNA gene amplicon sequencing was used to measure diversity of semen and fecal microbiomes. Total sperm count, total motility, progressive motility, DNA integrity, lipid peroxidation and mitochondrial oxidative stress, biomarkers of sperm quality, were measured after each intervention. A general linear model was employed to analyse and compare microbiome diversity measures and sperm quality data across four time points. Shannon's diversity index (alpha-diversity), and evenness of semen and gastrointestinal microbiomes were significantly different (p<0.001). A trend was observed for prebiotic effects on the diversity indices of the GI microbiome (p= 0.07). No effects of treatments were observed on either semen microbiome or sperm quality. Pre-, pro- and synbiotic supplements showed no negative effect on sperm quality parameters observed. This proof of concept provides preliminary data to inform future studies exploring the relationship between microbiomes and fertility.

Associations between Sperm Epigenetic Age and Semen Parameters: An Evaluation of Clinical and Non-Clinical Cohorts.

The well-documented relationship between chronological age and the sperm methylome has allowed for the construction of epigenetic clocks that estimate the biological age of sperm based on DNA methylation, which we previously termed sperm epigenetic age (SEA). Our lab demonstrated that SEA is positively associated with the time taken to achieve pregnancy; however, its relationship with semen parameters is unknown. A total of 379 men from the Longitudinal Investigation of Fertility and Environment (LIFE) study, a non-clinical cohort, and 192 men seeking fertility treatment from the Sperm Environmental Epigenetics and Development Study (SEEDS) were included in the study. Semen analyses were conducted for both cohorts, and SEA was previously generated using a machine learning algorithm and DNA methylation array data. Association analyses were conducted via multivariable linear regression models adjusting for BMI and smoking status. We found that SEA was not associated with standard semen characteristics in SEEDS and LIFE cohorts. However, SEA was significantly associated with higher sperm head length and perimeter, the presence of pyriform and tapered sperm, and lower sperm elongation factor in the LIFE study (p < 0.05). Based on our results, SEA is mostly associated with defects in sperm head morphological factors that are less commonly evaluated during male infertility assessments. SEA shows promise to be an independent biomarker of sperm quality to assess male fecundity.

Small RNAs: an ideal choice as sperm quality biomarkers.

Spermatozoa were classically known as vehicles for the delivery of the paternal genome to the oocyte. However, in 1962, spermatozoa were discovered to carry significant amounts of RNA in them, which raised questions about the significance of these molecules in such a highly specialized cell. Scientific research in the last six decades has investigated the biological significance of sperm RNAs by various means. Irrespective of what sperm RNAs do, their presence in spermatozoa has attracted attention for their exploitation as biomarkers of fertility. Research in this direction started in the year 2000 and is still underway. A major hurdle in this research is the definition of the standard human sperm RNAome. Only a few normozoospermic samples have been analyzed to define the normal sperm RNAome. In this article, we provide a perspective on the suitability of sperm RNAs as biomarkers of fertility and the importance of defining the normal sperm RNAome before we can succeed in identifying RNA-based biomarkers of sperm quality and fertility. The identification of sperm RNA biomarkers of fertility can be exploited for quality screening of donor sperm samples, explain infertility in idiopathic cases, and RNA therapeutics for the treatment of male infertility.

60 Molecular Characterization of Boar Semen in Relation to Sperm Quality

Abstract Artificial insemination (AI) with fresh semen is the primary breeding tool in the swine industry. However, the heterogenous semen doses create challenges in boar studs due to the lack of consistent assessment criteria. Seminal plasma (SP) surrounding spermatozoa is a great source of reliable non-invasive biomarkers of sperm quality. It contains a wide array of bioactive molecules, whose dynamic compositions in response to extrinsic and intrinsic factors influence sperm motility and morphology. Therefore, we hypothesize that the miRNA content of extracellular vesicles (EVs) found in SP may reflect semen quality. Our study aimed to characterize poor vs. good quality semen and compare miRNA profiles of SP-derived EVs. Raw semen was collected (n = 75) from sexually mature Duroc boars at a commercial stud (Prestage Farms, MS) for 8 weeks. Semen samples were subjected to sperm motility and morphology analyses and subsequently classified as poor (n = 38) or good (n = 37) quality based on assessment criteria cut-offs (&amp;lt; 70% and ≥70%, respectively). Thereafter, raw semen samples were subjected to serial centrifugation to collect spermatozoa and SP. Samples were stored at -80oC until analyses. A subset of each frozen-thawed SP was used for NIRS absorbance analysis (ASD FieldSpec3 portable spectrometer – 1mm quartz cuvettes; 750-2500 nm). Other subsets of frozen-thawed SP were ultra-centrifugated to isolate EVs (SP-EV), followed by confirmations through transmission electron microscopy (TEM), dynamic light scattering (DLS), and immuno-western blotting. Five pools of three individual SP-EV samples, corresponding to extremely poor and extremely good quality semen, were constituted for total exosome RNA isolation, small RNA sequencing, miRNA detection, and differential miRNA expression. Messenger RNA targets of miRNA were searched and subjected to functional bioinformatic analyses using various software algorithms. Significant differences were set for P &amp;lt; 0.05. Poor and good quality samples had different spectrum profiles following NIRS analyses. Changes were explicitly observed in water structures at coordinates C1, C5, and C12 (nm), corresponding to water associations with chaotropic solutes (water asymmetrical stretching vibrations-ν3), free water, and kosmotropic solutes (increased H-bonding numbers and strength), respectively. The NIRS-LDA data contrasted two distinctive poor and good quality SP samples. All boar SP-EV were confirmed: round spherical structure; 30 to 400nm of diameter; and CD9+, CD63+, and CD81+ membrane markers. Totals of 281 and 271 miRNAs were identified in poor and good groups, respectively, and 15 were differentially expressed (DE). Only three DE miRNAs (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) permitted targeting of 109 mRNA, which functional analyses indicated associations with nuclear and cytosol localization, as well as with functions, i.e., acetylation, Ubl conjugation, protein kinase binding, potentially altering sperm function. In conclusion, the study indicates that the NIRS tool can distinguish between good and poor semen quality, and the miRNA SP-EV content may reflect semen quality.

Regional abundances of binder of sperm (BSP) proteins are negatively associated with the quality of frozen-thawed bovine spermatozoa.

The localization and abundance of the sperm BSP proteins correlate with in vitro fertility in domestic bulls used in artificial insemination service. Binder of sperm (BSP) proteins, secreted mainly by the accessory sex glands, are the major protein family present in bovine seminal plasma and on the sperm surface after ejaculation. In vivo, BSP proteins facilitate sperm capacitation and sperm reservoir formation; however, their impact on sperm function within the in vitro systems is less clear. Therefore, this biomarker-based study aimed to characterize the localization and abundance of BSP proteins from in vitro processed frozen-thawed bovine spermatozoa. Using image-based flow cytometry and Western blotting, BSP protein localization, abundance, membrane and acrosomal integrity were investigated in the supernatant (nonmotile) and pellet (motile) fractions of gradient-separated bull spermatozoa. Spermatozoa from the supernatant fraction had high enrichment of all BSP proteins investigated (BSP1, BSP3, BSP5; P < 0.05) when compared to the pellet fraction. In the pellet fraction, BSP1 and BSP3 bound predominately to the acrosomal region, whereas BSP5 had a high affinity for the midpiece. However, in the supernatant fraction, BSP proteins predominately coated the entire sperm surface resulting in the loss of regional specificity. High BSP protein abundance in the spermatozoa also correlated with acrosome and membrane damage. Whereas a high abundance of BSP5 correlated with low embryo cleavage rates, high abundance of BSP1 on the sperm head coincided with a high blastocyst rate. Therefore, changes in the quantity and localization of specific BSP proteins could act as potential biomarkers of sperm quality and fertility.

Molecular biomarkers for sperm quality in an economically important fish: Blue catfish, Ictalurus furcatus

Intraspecies variability in sperm quality is common among aquaculture species, thus necessitating an urgent need to establish reliable molecular biomarkers to screen for males with high-quality sperm. The channel catfish (Ictalurus punctatus) ♀ × blue catfish (I. furcatus) ♂ hybrid accounts for >50% of United States catfish production. Blue catfish sperm cannot be collected using non-lethal methods, and thus, males must be sacrificed to determine reproductive quality. To establish gene expression biomarkers of sperm quality, we characterized important sperm traits, ranked blue catfish males based on the curvilinear velocity of their sperm, and sequenced testicular tissue for mRNAs and sRNAs. Of the 43 males characterized, testicular tissue from 4 individuals with low-velocity (155.70 mm/s ± 1.49 SD) and 4 with high-velocity (205.01 mm/s ± 4.63 SD) were chosen for sequencing. No differentially expressed sRNAs were detected between the two sperm performance groups. However, we identified two differentially expressed mRNA transcripts between the low-velocity and high-velocity groups, aqp3a and ly97.3, both upregulated in the high-velocity group compared to the low-velocity group. These molecular biomarkers of sperm quality in testicular tissue are linked to gamete quality and may be applied for more efficient catfish aquaculture.

The development of new biomarkers of spermatozoa quality in cattle.

There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high (n = 4) or low (n = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater (p < 0.05) incidence of aggresome defects than post-gradient samples from high-performing sires. Additionally, IBFC was used to evaluate spermatozoa viability following gradient purification. Distinct populations of sperm cells were identified. High-performing sires had more spermatozoa in the population deemed most viable than low-performing sires. This study demonstrated that spermatozoa defects vary in populations before and following gradient purification, indicating that it may be beneficial to separately evaluate semen for in vivo and in vitro use. Furthermore, a prevalent defect in low-performing sires that could explain a discrepancy between successful fertilization and embryo development was identified. Therefore, elucidating a malfunction regulated by sire, that could potentially affect early embryo development.

Is telomere length a biomarker of sperm quality? A systematic review and meta-analysis of observational studies.

Telomeres are essential for the integrity of chromosome ends during cell division and their involvement in different processes linked to aging has been established. These chromosome components are involved in spermatogenesis and seem to play an important role in fertilization and embryo development. Telomere length is shortened with each cell division. Recently, short sperm telomere length has been proposed as a potential biomarker of male infertility. To conduct a systematic review and meta-analysis of studies exploring the association between spermatozoa and/or leukocyte telomere length with sperm quality parameters and different infertility conditions. A systematic review and meta-analysis was conducted with studies from Medline-PUBMED and Cochrane Library databases until May 2022. Eligible studies included cohort, cross-sectional and case-control studies, and telomere length in spermatozoa and/or leukocytes cells was defined as the exposure. Semen quality parameters or infertility conditions (e.g., oligozoospermia, asthenozoospermia, teratozoospermia, or other spermatogenic impairment combinations) were defined as the outcomes. Twenty-three observational studies were included. In the qualitative analysis, high heterogeneity was observed between studies regarding the associations between telomere length and semen parameters in different normozoospermic/fertile and oligozoospermic/infertile populations. In the meta-analysis, spermatozoa and leukocyte telomere length were shorter in infertile individuals than in fertile individuals (mean difference [95% confidence interval]: -1.43 [-1.66 to -1.21], p-value <0.001and -1.67 [-2.02 to -1.31], p-value <0.001, respectively). Moreover, in terms of sperm telomere length, these differences were also significant between individuals with a normal seminogram and individuals with a low quantity of spermatozoa in the ejaculate (-0.97 [-1.32, -0.61], p-value <0.001). The current systematic review and meta-analysis suggests the potential role of spermatozoa or leukocyte telomere length as a reliable biomarker of semen quality, which may help distinguish between infertility conditions beyond the routine semen analysis.

Telomere Length, a New Biomarker of Male (in)Fertility? A Systematic Review of the Literature.

Male factors are suspected in around half cases of infertility, of which up to 40% are diagnosed as idiopathic. In the context of a continuously increased resort to ART and increased decline of semen parameters, it is of greatest interest to evaluate an additional potential biomarker of sperm quality. According to PRISMA guidelines, this systematic review of the literature selected studies evaluating telomere length in sperm and/or in leukocytes as a potential male fertility biomarker. Twenty-two publications (3168 participants) were included in this review of experimental evidence. For each study, authors determined if there was a correlation between telomere length and semen parameters or fertility outcomes. Of the 13 studies concerning sperm telomere length (STL) and semen parameters, ten found an association between short STL and altered parameters. Concerning the impact of STL on ART results, the data are conflicting. However, eight of the 13 included studies about fertility found significantly longer sperm telomeres in fertile men than in infertile men. In leukocytes, the seven studies reported conflicting findings. Shorter sperm telomeres appear to be associated with altered semen parameters or male infertility. Telomere length may be considered as a new molecular marker of spermatogenesis and sperm quality, and thus is related to male fertility potential. However, additional studies are needed to define the place of the STL in the assessment of individual fertility.

Characterization of Extracellular Vesicle-Coupled miRNA Profiles in Seminal Plasma of Boars with Divergent Semen Quality Status.

Sperm heterogeneity creates challenges for successful artificial insemination. Seminal plasma (SP) surrounding sperm is an excellent source for detecting reliable non-invasive biomarkers of sperm quality. Here, we isolated microRNAs (miRNAs) from SP-derived extracellular vesicles (SP-EV) of boars with divergent sperm quality statuses. Raw semen from sexually mature boars was collected for eight weeks. Sperm motility and normal morphology were analyzed, and the sperm was classified as poor- or good-quality based on standard cutoffs of 70% for the parameters measured. SP-EVs were isolated by ultracentrifugation and confirmed by electron microscopy, dynamic light scattering, and Western immunoblotting. The SP-EVs were subjected to total exosome RNA isolation, miRNA sequencing, and bioinformatics analysis. The isolated SP-EVs were round spherical structures approximately 30-400 nm in diameter expressing specific molecular markers. miRNAs were detected in both poor- (n = 281) and good (n = 271)-quality sperm, with fifteen being differentially expressed. Only three (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) allowed gene targeting associated with cellular localization (nuclear and cytosol) and molecular functions (acetylation, Ubl conjugation, and protein kinase binding), potentially impairing sperm quality. PTEN and YWHAZ emerged as essential proteins for protein kinase binding. We conclude that SP-EV-derived miRNAs reflect boar sperm quality to enable therapeutic strategies to improve fertility.

Effects of activation and assisted reproduction techniques on the composition, structure, and properties of the sauger (Sander Canadensis) spermatozoa plasma membrane

The sperm plasma membrane is a multifunctional organelle essential to fertilization. However, assisted reproduction techniques often negatively affect this structure, resulting in reduced fertility. These reductions have been attributed to plasma membrane damage in a wide array of species, including fish. Considerable research has been conducted on the fish sperm membrane, but few have examined the effect of cryopreservation and other assisted reproduction techniques (ARTs) on not only membrane composition, but also specific characteristics (e.g., fluidity) and organization (e.g., lipid rafts). Herein, we determined the effects of three ARTs (testicular harvest, strip spawning, and cryopreservation) on the sperm plasma membrane, using Sauger (Sander canadensis) sperm as a model. To this end, a combination of fluorescent dyes (e.g., merocyanine 540, filipin III, cholera toxin subunit β), liquid chromatography – mass spectroscopy (LC-MS) analysis of membrane lipids, and membrane ultracentrifugation coupled with plate assays and immunofluorescence were used to describe and compare sperm fluidity, membrane composition, as well as lipid raft composition and distribution among sperm types. Stripped sperm became more fluid following motility activation (40% increase in highly fluid cells characterized by a 2 × increase in fluorescence) and contained lipid rafts restricted to the midpiece. Testicular harvest yielded sperm with characteristics similar to stripped sperm. By contrast, cryopreservation impacted every aspect of membrane physiology. Two cell populations, one highly fluid and the other rigid, resulted from the freeze-thaw process. Cryopreservation reduced lipid raft cholesterol content by 44% and flotilin-2 (a lipid raft marker) was partially displaced owing to a decrease in buoyancy. Unlike stripped and testicular sperm, LC-MS analysis revealed increases in oxidative damage markers, membrane destabilization, and apoptotic signaling in cryopreserved sperm. Ultrastructural analysis also revealed widespread physical damage to the membrane following freeze-thaw. Sperm motility, however, was unrelated to any measure of membrane physiology used in this study. Our results demonstrate that ARTs have the potential to substantially affect the sperm plasma membrane, but not always detrimentally. These results provide multiple potential biomarkers of sperm quality as well as insight into sources of sub-fertility resulting from use of ARTs.

The proAKAP4 concentrations in Nelore bull sperm and their relation to FTAI conception rate results

The AKAP4 protein has an essential role in sperm motility, and its precursor, the proAKAP4, is considered a biomarker for sperm quality and fertility. Despite this, proAKAP4 concentration was never evaluated in Bos indicus sperm. Therefore, this study aims to determine the proAKAP4 concentration in the sperm of Nellore bulls and its association with sperm kinetics, morpho-functionality, morphology, and conception rates after fixed-time artificial insemination (FTAI). The bulls (n = 9) used in our study presented sperm characteristics above expected standards for quality. Sperm from each bull was evaluated regarding kinetics by the CASA system, and assessed for mitochondrial functionality, sperm membrane integrity, and morphology. For the FTAI protocol, multiparous Nelore cows (n = 1507) were inseminated with frozen-thawed sperm from the same batches used in the laboratory analysis. Additionally, the sperm proAKAP4 concentration was determined using an ELISA kit. The data were analyzed by linear regression and the significance level was set to 5%. The mean conception rate was 52.52%. The mean proAKAP4 concentration was 25.75 ng/106 spermatozoa. The proAKAP4 concentration positively affected the sperm total and progressive motilities, and conception rates after FTAI (respectively, r2 = 0.49, P = 0.03; r2 = 0.63, P = 0.01; and r2 = 0.51, P = 0.03). Based on the relationship detected in this study, we conclude that the sperm proAKAP4 concentration is a suitable biomarker to predict the quality and fertility of frozen-thawed Bos indicus semen.

Revisiting the impact of varicocele and its treatments on male fertility

Varicocele is one of the most common, yet treatable, causes of male infertility. Varicoceles are present in more than 40% of infertile men with primary infertility, a figure that increases with age. Varicoceles impair semen parameters and sperm DNA and are linked with lower pregnancy and live birth rates. Until recently, men had seldom been examined in male fertility workups. This is changing as urologists have become recognized as team members in infertility. Hence identification and treatment are available as never before. Furthermore, as men become aware that they are as likely as their female partners to be infertile, they want equal ‘couple care’, requesting urological referrals as they realize that they can improve their semen quality and chances of fatherhood without or before fertility treatment. There is now a greater understanding of the mechanisms of varicocele-induced damage by oxidative stress, using sperm DNA as a sensitive biomarker of sperm quality. There is a current consensus that varicocele is linked to poor semen and repair improves semen and sperm DNA quality. Evidence is strengthening to indicate that varicocele repair increases pregnancy and live birth rates in natural conception and following fertility treatment.

P-049 Sperm GSTM3: a potential molecular biomarker for sperm quality and male (in)fertility

Abstract Study question Is sperm Glutathione S-transferase Mu 3 (GSTM3) associated to sperm physiological status and male (in)fertility? Summary answer GSTM3 is expressed in human sperm, and is associated to sperm quality and fertility, which suggests it as a potential molecular biomarker for male (in)fertility. What is known already GSTM3 is an antioxidant enzyme expressed in sperm cells that is essential for an appropriate mitochondrial function, plasma membrane stability and oxidative regulation. Previous evidence suggested sperm GSTM3 to be useful as an infertility prognosis and diagnosis tool in mammalian species. In humans, expression of sperm GSTM3 has been found to be altered in mitochondrial-impaired sperm and in sperm from patients with unilateral varicocele. However, neither has this enzyme been related to semen alterations nor has it been reported to be associated to male infertility. Study design, size, duration A total of 34 semen samples from healthy, fertile men and men referred for infertility evaluation were collected, analysed and subsequently cryopreserved. Samples were classified as fertile having normal sperm parameters (NSP; n = 10); infertile with asthenoteratozoospermia (AT; n = 8) or oligoasthenoteratozoospermia (OAT; n = 8); or with idiopathic infertility (n = 8). Finally, frozen-thawed sperm samples were assessed for DNA fragmentation, and the presence, localization and relative content of GSTM3. Protein expression of GSTM3 in sperm was compared between groupss. Participants/materials, setting, methods All samples were initially evaluated by conventional sperm analysis (ejaculate volume and pH, sperm concentration, motility and morphology). Protein expression profile of GSTM3 was determined through immunoblotting and immunolocalization assays. Sperm GSTM3 was quantified using an enzyme-linked immunosorbent assay. Spearman's rank-order correlation coefficients between GSTM3 levels and sperm quality parameters were calculated. Statistical differences between groups were determined by Kruskal-Wallis followed by Mann-Whitney U tests. Main results and the role of chance GSTM3 was found to be present in human sperm cells, and to be localized along the tail. Sperm GSTM3 was positively and significantly correlated with sperm quality parameters (concentration [Rs=0.51], morphology [Rs=0.42], total [Rs=0.60] and progressive motility [Rs=0.58]; P &amp;lt; 0.05). Sperm GSTM3 levels were compared between sperm quality groups (NSP, AT and OAT). Sperm GSTM3 levels were found to be significantly higher (P &amp;lt; 0.05) in men with NSP than in those with AT or OAT (62.1, 28.1 and 20.6 ng GSTM3/mg total protein, respectively). Finally, levels of GSTM3 in sperm were compared between fertile and infertile men. Whereas fertile and idiopathic infertile men showed similar sperm GSTM3 levels (63.7 vs. 60.0 ng GSTM3/mg total protein, respectively, P &amp;gt; 0.05), infertile men with an altered seminogram had significantly lower (P &amp;lt; 0.05) levels (24.4 ng GSTM3/mg total protein). Limitations, reasons for caution The limited sample size of the present study warrants further research and clinical trials with larger sample sets before implementing this biomarker as a molecular diagnostic tool in fertility clinics. Wider implications of the findings The use of sperm GSTM3 as a novel biomarker in fertility clinics may be translated into cost-effective, non-invasive, time-saving and accurate diagnosis of men (in)fertility. Trial registration number N/A

Protamines and DNA integrity as a biomarkers of sperm quality and assisted conception outcome.

Present research aim was to identify functional tests in semen associated with DNA damage and chromatin maturity (protamination) which predict the outcome in assisted reproduction. Couples were grouped according to male partner semen parameters, into normozoospermia (NZs), severe male factor (SMF) and mild male factor (MMF). DNA fragmentation index (DFI) in spermatozoa was analysed by sperms chromatin dispersion (SCD), sperm chromatin structure assay (SCSA) and acridine orange testing (AOT). Chromomycin A3 (CMA3) and toluidine blue (TB) staining to measure sperm chromatin maturity (CM). DFI and chromatin decondensation were significantly lower in N compared to male factor categories (MMF and SMF). Aneuploidy embryos were significantly higher in couples with male factor infertility (MMF and SMF). A positive correlation was observed between fertilization rate (FR) and live birth rate (LBR) with sperm concentration, motility, vitality, normal sperm morphology and negative correlation between sperm DFI and sperm CM. No correlation was observed between embryo aneuploidy and sperm DFI or CM. Lower percentage of spermatozoa chromatin integrity are associated with low fertilization and live birth rate. Male factor infertility, due to impaired semen parameters and chromatin defects could be regarded in future as an indication of IVF/ICSI, and predictor of assisted reproductive techniques outcome.

Sperm DNA 5-methyl cytosine and RNA N6-methyladenosine methylation are differently affected during periods of body weight losses and body weight gain of young and mature breeding bulls.

Aiming to characterize the effects of nutritional status on epigenetic markers, such as DNA 5-methyl cytosine (mC) methylation and RNA N6-methyladenosine (m6A) methylation, of bovine sperm, 12 Angus × Hereford crossbred breeding bulls were submitted to nutritional changes for a period of 180 d: no change in body weight (BW) (phase 1 = 12 d), BW loss (phase 2 = 78 d), and BW gain (phase 3 = 90 d) in a repeated measures design. Animals were fed Beardless wheat (Triticum aestivum) hay and mineral mix. Statistical analyses were performed using SAS 9.4 (SAS Inst., Cary, NC). Higher levels of RNA m6A (P = 0.004) and DNA methylation (P = 0.007) of spermatic cells were observed at phase 2 compared with phase 1. In phase 3, sperm RNA m6A methylation levels continued to be higher (P = 0.004), whereas the DNA of sperm cells was similar (P = 0.426) compared with phase 1. Growing bulls had a tendency (P = 0.109) of higher RNA m6A methylation levels than mature bulls. Phase 2 altered scrotal circumference (P < 0.001), sperm volume (P = 0.007), sperm total motility (P = 0.004), sperm progressive motility (P = 0.004), total sperm count (P = 0.049), normal sperm (P < 0.001), abnormal sperm (P < 0.001), primary sperm defects (P = 0.039), and secondary sperm defects (P < 0.001). In phase 3, bulls had scrotal circumference, sperm volume, sperm motility, sperm progressive motility, total sperm count, normal and abnormal spermatozoa, and primary and secondary spermatozoa defects similar to phase 1 (P > 0.05). Serum concentrations of insulin-like growth factor-1 and leptin decreased during phase 2 (P = 0.010), while no differences (P > 0.05) were detected between phases 3 and 1; growing bulls tended (P = 0.102) to present higher leptin levels than mature bulls. Specific for mature bulls, DNA methylation was positively correlated with leptin concentration (0.569, P = 0.021), whereas for young bulls, DNA methylation was positively correlated with abnormal spermatozoa (0.824, P = 0.006), primary spermatozoa defect (0.711, P = 0.032), and secondary spermatozoa defect (0.661, P = 0.052) and negatively correlated with normal spermatozoa (-0.824, P = 0.006), total sperm count (-0.702, P = 0.035), and sperm concentration (-0.846, P = 0.004). There was no significant correlation (P > 0.05) between RNA m6A and hormones and semen traits. In conclusion, the nutritional status of breeding bulls alters epigenetic markers, such as DNA methylation and RNA m6A methylation, in sperm, and the impact of change seems to be age dependent. These markers may serve as biomarkers of sperm quality and fertility of bulls in the future. Detrimental effects on sperm production and seminal quality are observed at periods and places when and where environmental and nutritional limitations are a year-round reality and may carry hidden players that may influence a lifetime of underperformance.

Direct but Not Indirect Methods Correlate the Percentages of Sperm With Altered Chromatin to the Intensity of Chromatin Damage.

Although sperm chromatin damage, understood as damage to DNA or affectations in sperm protamination, has been proposed as a biomarker for sperm quality in both humans and livestock, the low incidence found in some animals raises concerns about its potential value. In this context, as separate methods measure different facets of chromatin damage, their comparison is of vital importance. This work aims at analyzing eight techniques assessing chromatin damage in pig sperm. With this purpose, cryopreserved sperm samples from 16 boars were evaluated through the following assays: TUNEL, TUNEL with decondensation, SCSA, alkaline and neutral sperm chromatin dispersion (SCD) tests, alkaline and neutral Comet assays, and chromomycin A3 test (CMA3). In all cases, the extent of chromatin damage and the percentage of sperm with fragmented DNA were determined. The degree of chromatin damage and the percentage of sperm with fragmented DNA were significantly correlated (p < 0.05) in direct methods (TUNEL, TUNEL with decondensation, and alkaline and neutral Comet) and CMA3, but not in the indirect ones (SCD and SCSA). Percentages of sperm with fragmented DNA determined by alkaline Comet were significantly (p < 0.05) correlated with TUNEL following decondensation and CMA3; those determined by neutral Comet were correlated with the percentage of High DNA Stainability (SCSA); those determined by SCSA were correlated with neutral and alkaline SCD; and those determined by neutral SCD were correlated with alkaline SCD. While, in pigs, percentages of sperm with fragmented DNA are directly related to the extent of chromatin damage when direct methods are used, this is not the case for indirect techniques. Thus, the results obtained herein differ from those reported for humans in which TUNEL, SCSA, alkaline SCD, and alkaline Comet were found to be correlated. These findings may shed some light on the interpretation of these tests and provide some clues for the standardization of chromatin damage methods.

Evaluation of bovine sperm telomere length and association with semen quality

The study aimed to evaluate if the sperm telomere length can be considered as a new biomarker for sperm quality in bulls. Sperm Telomere Length was evaluated by Monochrome Multiplex Quantitative PCR in group A (n = 8) and group B (n = 8) bulls, classified according to standard semen analysis. Also, this parameter was measured before and after Percoll gradient separation within bulls that produced semen of satisfactory quality. Sperm telomere length, measured as T/S ratio (average ratio of telomere repeats copy number to a single copy gene), was higher in group A than in group B bulls (0.77 ± 0.03 vs 0.43 ± 0.06; P < 0.01). Sperm telomere length was positively correlated with motility, viability and membrane integrity, and it was negatively correlated with sperm anomalies. Furthermore, Percoll gradient selected sperms with higher T/S ratio than unselected sperms (1.19 ± 0.02 vs 0.67 ± 0.03). These results suggest that sperm telomere length can be used as a new marker of bovine semen quality.