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Purpose: Sperm DNA fragmentation (SDF) is a functional sperm abnormality that can impact reproductive potential, for which four assays have been described in the recently published sixth edition of the WHO laboratory manual for the examination and processing of human semen. The purpose of this study was to examine the global practices related to the use of SDF assays and investigate the barriers and limitations that clinicians face in incorporating these tests into their practice., Materials and Methods: Clinicians managing male infertility were invited to complete an online survey on practices related to SDF diagnostic and treatment approaches. Their responses related to the technical aspects of SDF testing, current professional society guidelines, and the literature were used to generate expert recommendations via the Delphi method. Finally, challenges related to SDF that the clinicians encounter in their daily practice were captured., Results: The survey was completed by 436 reproductive clinicians. Overall, terminal deoxynucleotidyl transferase deoxyuridine triphosphate Nick-End Labeling (TUNEL) is the most commonly used assay chosen by 28.6%, followed by the sperm chromatin structure assay (24.1%), and the sperm chromatin dispersion (19.1%). The choice of the assay was largely influenced by availability (70% of respondents). A threshold of 30% was the most selected cut-off value for elevated SDF by 33.7% of clinicians. Of respondents, 53.6% recommend SDF testing after 3 to 5 days of abstinence. Although 75.3% believe SDF testing can provide an explanation for many unknown causes of infertility, the main limiting factors selected by respondents are a lack of professional society guideline recommendations (62.7%) and an absence of globally accepted references for SDF interpretation (50.3%)., Conclusions: This study represents the largest global survey on the technical aspects of SDF testing as well as the barriers encountered by clinicians. Unified global recommendations regarding clinician implementation and standard laboratory interpretation of SDF testing are crucial., Competing Interests: The authors have nothing to disclose., (Copyright © 2024 Korean Society for Sexual Medicine and Andrology.)

Purpose: Sperm DNA fragmentation (SDF) has been associated with male infertility and poor outcomes of assisted reproductive technology (ART). The purpose of this study was to investigate global practices related to the management of elevated SDF in infertile men, summarize the relevant professional society recommendations, and provide expert recommendations for managing this condition., Materials and Methods: An online global survey on clinical practices related to SDF was disseminated to reproductive clinicians, according to the CHERRIES checklist criteria. Management protocols for various conditions associated with SDF were captured and compared to the relevant recommendations in professional society guidelines and the appropriate available evidence. Expert recommendations and consensus on the management of infertile men with elevated SDF were then formulated and adapted using the Delphi method., Results: A total of 436 experts from 55 different countries submitted responses. As an initial approach, 79.1% of reproductive experts recommend lifestyle modifications for infertile men with elevated SDF, and 76.9% prescribe empiric antioxidants. Regarding antioxidant duration, 39.3% recommend 4-6 months and 38.1% recommend 3 months. For men with unexplained or idiopathic infertility, and couples experiencing recurrent miscarriages associated with elevated SDF, most respondents refer to ART 6 months after failure of conservative and empiric medical management. Infertile men with clinical varicocele, normal conventional semen parameters, and elevated SDF are offered varicocele repair immediately after diagnosis by 31.4%, and after failure of antioxidants and conservative measures by 40.9%. Sperm selection techniques and testicular sperm extraction are also management options for couples undergoing ART. For most questions, heterogenous practices were demonstrated., Conclusions: This paper presents the results of a large global survey on the management of infertile men with elevated SDF and reveals a lack of consensus among clinicians. Furthermore, it demonstrates the scarcity of professional society guidelines in this regard and attempts to highlight the relevant evidence. Expert recommendations are proposed to help guide clinicians., Competing Interests: The authors have nothing to disclose., (Copyright © 2023 Korean Society for Sexual Medicine and Andrology.)

Purpose: Sperm DNA fragmentation (SDF) testing was recently added to the sixth edition of the World Health Organization laboratory manual for the examination and processing of human semen. Many conditions and risk factors have been associated with elevated SDF; therefore, it is important to identify the population of infertile men who might benefit from this test. The purpose of this study was to investigate global practices related to indications for SDF testing, compare the relevant professional society guideline recommendations, and provide expert recommendations., Materials and Methods: Clinicians managing male infertility were invited to take part in a global online survey on SDF clinical practices. This was conducted following the CHERRIES checklist criteria. The responses were compared to professional society guideline recommendations related to SDF and the appropriate available evidence. Expert recommendations on indications for SDF testing were then formulated, and the Delphi method was used to reach consensus., Results: The survey was completed by 436 experts from 55 countries. Almost 75% of respondents test for SDF in all or some men with unexplained or idiopathic infertility, 39% order it routinely in the work-up of recurrent pregnancy loss (RPL), and 62.2% investigate SDF in smokers. While 47% of reproductive urologists test SDF to support the decision for varicocele repair surgery when conventional semen parameters are normal, significantly fewer general urologists (23%; p=0.008) do the same. Nearly 70% would assess SDF before assisted reproductive technologies (ART), either always or for certain conditions. Recurrent ART failure is a common indication for SDF testing. Very few society recommendations were found regarding SDF testing., Conclusions: This article presents the largest global survey on the indications for SDF testing in infertile men, and demonstrates diverse practices. Furthermore, it highlights the paucity of professional society guideline recommendations. Expert recommendations are proposed to help guide clinicians., Competing Interests: The authors have nothing to disclose., (Copyright © 2023 Korean Society for Sexual Medicine and Andrology.)

The global human population is exponentially increasing, which requires the production of quality food through efficient reproduction as well as sustainable production of livestock. Lack of knowledge and technology for assessing semen quality and predicting bull fertility is hindering advances in animal science and food animal production and causing millions of dollars of economic losses annually. The intent of this systemic review is to summarize methods from computational biology for analysis of gene, metabolite, and protein networks to identify potential markers that can be applied to improve livestock reproduction, with a focus on bull fertility. We provide examples of available gene, metabolic, and protein networks and computational biology methods to show how the interactions between genes, proteins, and metabolites together drive the complex process of spermatogenesis and regulate fertility in animals. We demonstrate the use of the National Center for Biotechnology Information (NCBI) and Ensembl for finding gene sequences, and then use them to create and understand gene, protein and metabolite networks for sperm associated factors to elucidate global cellular processes in sperm. This study highlights the value of mapping complex biological pathways among livestock and potential for conducting studies on promoting livestock improvement for global food security.

To investigate the effects of culture media with different lactate concentrations on early embryonic development, data collected from our patients undergoing preimplantation genetic testing (PGT) were assessed using the EmbryoScope™ time-lapse culturing system. After intracytoplasmic sperm injection (ICSI), sibling oocytes were cultured in the same EmbryoScope (Vitrolife) slides including two different commercially available media. The patients with fewer than five mature oocytes were not included in the analyses. All embryos were hatched on day 3, and trophectoderm biopsies (n = 212) were performed accordingly. PGT for aneuploidy (PGT-A) on biopsied materials was carried out using next generation sequencing. Morphokinetic parameters, fertilization, irregular division, degeneration, blastulation, euploidy, and pregnancy rates of embryos cultured in LifeGlobal Global Total medium (LGGT) and Continuous Single Culture-NX Complete medium (CSCM-NXC) were compared. There were no differences observed in time to pronuclear fade, or in time spent as 2-cell (cc2) and 3-cell (s2), to 4-cell, 5-cell, morula and blastocyst stages (P > 0.05). Embryos reached the 2-cell (t2) and 3-cell (t3) stages significantly faster in LGGT (P < 0.05), whereas embryos grown in CSCM-NXC with lower lactate reached starting blastulation significantly sooner (P = 0.026). However, there were no statistical differences observed in fertilization, blastulation, degeneration, irregular division euploidy, and pregnancy rates between the two groups (P > 0.05). Even though pregnancy and fertilization rates did not indicate statistical differences, results are significant to provide better insight on potential roles of lactate in embryo development. These finding will advance the fundamental knowledge of human embryo development and assisted reproductive technologies.

Prediction of bull fertility is critical for the sustainability of both dairy and beef cattle production. Even though bulls produce ample amounts of sperm with normal parameters, some bulls may still suffer from subpar fertility. This causes major economic losses in the cattle industry because using artificial insemination, semen from one single bull can be used to inseminate hundreds of thousands of cows. Although there are several traditional methods to estimate bull fertility, such methods are not sufficient to explain and accurately predict the subfertility of individual bulls. Since fertility is a complex trait influenced by a number of factors including genetics, epigenetics, and environment, there is an urgent need for a comprehensive methodological approach to clarify uncertainty in male subfertility. The present review focuses on molecular and functional signatures of bull sperm associated with fertility. Potential roles of functional genomics (proteome, small noncoding RNAs, lipidome, metabolome) on determining male fertility and its potential as a fertility biomarker are discussed. This review provides a better understanding of the molecular signatures of viable and fertile sperm cells and their potential to be used as fertility biomarkers. This information will help uncover the underlying reasons for idiopathic subfertility., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

Developing a deeper understanding of biological components of sperm is essential to improving cryopreservation techniques and reproductive technologies. To fully ascertain the functional determinants of fertility, lipidomic methods have come to the forefront. Lipidomics is the study of the lipid profile (lipidome) within a cell, tissue, or organism and provides a quantitative analysis of the lipid content in that sample. Sperm cells are composed of various lipids, each with their unique contribution to the overall function of the cell. Lipidomics has already been used to find new and exciting information regarding the fatty acid content of sperm cells from different species. While the applications of lipidomics are rapidly evolving, gaps in the knowledge base remain unresolved. Current limitations of lipidomics studies include the number of available samples to analyze and the total amount of cells within those samples needed to detect changes in the lipid profiles across different subjects. The information obtained through lipidomics research is essential to systems and cellular biology. This review provides a concise analysis of the most recent developments in lipidomic research. This scientific resource is important because these developments can be used to not only combat the reproductive challenges faced when using cryopreserved semen and artificial reproductive technologies in livestock such as cattle, but also other mammals, such as humans or endangered species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Evans, Dinh, Hardcastle, Gilmore, Ugur, Hitit, Jousan, Nicodemus and Memili.)

The objective of the current study was to determine the fatty acid composition of sperm from Holstein bulls with different freezability (Good and Poor; n = 12). Fatty acids were extracted from frozen sperm in 1:2 (v/v) chloroform-methanol solvent, fractionated into neutral and polar fractions, and composition determined by gas chromatography-mass spectrometry. Thirty-four fatty acids were quantified and their concentrations and percentages within each lipid fraction were calculated. Overall, saturated fatty acids (SFA) were predominant, accounting for 71 to 80% of fatty acids in neutral and polar lipid factions. There were marked differences in fatty acid composition between the lipid fractions (P < 0.001). The branched chain fatty acid (BCFA) concentration (15 to 18 µg) was almost twice as much as polyunsaturated fatty acids (PUFA) concentration found in the polar lipid fraction (8 to 9 µg; P < 0.001). Sperm with different freezability phenotypes only had a few differences in 22:0, 18:1 cis 9, and 14:0 13-methyl fatty acids (P ≤ 0.011). These results are significant because they reveal key understandings of fatty acid composition of sperm membrane and lay a foundation for the manipulation of membrane integrity, fluidity, and stability to advance the assisted reproductive technologies.

The objective of this study was to ascertain the cellular and functional parameters as well as ROS related changes in sperm from bulls with varied sperm freezability phenotypes. Using principal component analysis (PCA), the variables were reduced to two principal components, of which PC1 explained 48% of the variance, and PC2 explained 24% of the variance, and clustered animals into two distinct groups of good freezability (GF) and poor freezability (PF). In ROS associated pathophysiology, there were more dead superoxide anion positive (Dead SO+) sperm in GF bulls than those in PF (15.72 and 12.00%; P = 0.024), and that Dead SO+ and live hydrogen positive cells (live H 2 O 2 +) were positively correlated with freezability, respectively ( R 2 = 0.55, P < 0.0130) and (r s = 0.63, P = 0.0498). Related to sperm functional integrity, sperm from PF bulls had greater dead intact acrosome (DIAC) than those from GF bulls (26.29 and 16.10%; P = 0.028) whereas sperm from GF bulls tended to have greater live intact acrosome (LIAC) than those from PF bulls (64.47 and 50.05%; P = 0.084). Sperm with dead reacted acrosome (DRAC) in PF bulls were greater compared to those in GF (19.27 and 11.48%; P = 0.007). While DIAC ( R 2 = 0.56, P = 0.0124) and DRAC ( R 2 = 0.57, P < 0.0111) were negatively correlated with freezability phenotype, LIAC ( R 2 = 0.36, P = 0.0628) was positively correlated. Protamine deficiency (PRM) was similar between sperm from GF and PF bulls (7.20 and 0.64%; P = 0.206) and (r s = 0.70, P = 0.0251) was correlated with freezability. Sperm characteristics associated with cryotolerance are important for advancing both fundamental andrology and assisted reproductive technologies across mammals., (Copyright © 2020 Hitit, Ugur, Dinh, Sajeev, Kaya, Topper, Tan and Memili.)

MicroRNAs modulate male fertility by regulating gene expression. In this study, dynamics of sperm miR-15a, miR-29b and miR-34a from high fertility (HF) and low fertility (LF) bulls using RT-qPCR were evaluated. Bioinformatic tools were employed to ascertain genes of interest of the sperm miRNAs. The expression levels of p53, BCL2, BAX and DNMT1 in bull spermatozoa were determined by immunoblotting. MicroRNA levels of miR-15a and miR-29 were higher in LF sires when compared with those present in HF bulls. Expression levels of miR-34a did not differ between the two groups. We found an inverse correlation between miR-15a and bull fertility. MiR29-b was also negatively associated with fertility scores. BCL2 and DNMT1 were higher in HF bulls while BAX was higher in the LF group. Our data showed a positive correlation between BCL2 and bull fertility. In addition, DNMT1 was positively associated with bull fertility. Furthermore, levels of BAX were negatively linked with bull fertility scores. Identification of miRNAs found in the spermatozoa of sires with different in vivo fertility helps understand the alterations in the fertilising capacity from cattle and other mammals. These potential biomarkers can be used in reproductive biotechnology as fertility markers to assess semen quality and predict male fertility., (© 2019 Blackwell Verlag GmbH.)

Sperm cryopreservation is an important technique for fertility management, but post-thaw viability of sperm differs among breeding bulls. With metabolites being the end products of various metabolic pathways, the contributions of seminal plasma metabolites to sperm cryopreservation are still unknown. These gaps in the knowledge base are concerning because they prevent advances in the fundamental science of cryobiology and improvement of bull fertility. The objective of this study was to test the hypothesis that seminal plasma amino acids are associated with freezability of bull sperm. To accomplish this objective, amino acid concentrations in seminal plasma from seven bulls of good freezability (GF) and six bulls of poor freezability (PF) were quantified using gas chromatography-mass spectrometry (GC-MS). Multivariate and univariate analyses were performed to identify potential freezability biomarkers. Pathways and networks analyses of identified amino acids were performed using bioinformatic tools. By analyzing and interpreting the results we demonstrated that glutamic acid was the most abundant amino acid in bull seminal plasma with average concentration of 3,366 ± 547.3 nM, which accounts for about 53% of total amino acids. The other most predominant amino acids were alanine, glycine, and aspartic acid with the mean concentrations of 1,053 ± 187.9, 429.8 ± 57.94, and 427 ± 101.3 nM. Pearson's correlation analysis suggested that phenylalanine concentration was significantly associated with post-thaw viability ( r = 0.57, P -value = 0.043). Significant correlations were also found among other amino acids. In addition, partial least squares-discriminant analysis (PLS-DA) bi-plot indicated a distinct separation between GF and PF groups. Phenylalanine had the highest VIP score and was more abundant in the GF groups than in the PF groups. Moreover, pathway and network analysis indicated that phenylalanine contributes to oxidoreductase and antioxidant reactions. Although univariate analysis did not yield significant differences in amino acid concentration between the two groups, these findings are significant that they indicate the potentially important roles of amino acids in seminal plasma, thereby building a foundation for the fundamental science of cryobiology and reproductive biotechnology., (Copyright © 2020 Ugur, Dinh, Hitit, Kaya, Topper, Didion and Memili.)

The objective of this study was to ascertain cellular characteristics and the dynamics of the sperm chromatin proteins protamine 1 (PRM1) and protamine 2 (PRM2) in the sperm of Holstein bulls having a different fertility status. Important sperm variables were analyzed using computer-assisted sperm analysis (CASA). Sperm membrane, acrosome status, DNA integrity were also assessed using propidium iodide (PI), fluorescein isothiocyanate conjugated to Arachis hypogaea (FITC-PNA), and acridine orange (AO) followed by flow cytometry. In addition, abundances of PRM1 and PRM2 were analyzed using flow cytometry experiments. Differences in sperm decondensation capacity were assessed in bulls of varying fertility using a decondensation assay. As determined using CASA, average pathway velocity, amplitude of lateral head displacement and straightness were different (P < 0.05) for sperm from high and low fertility bulls. There, however, were no differences between the high and low fertility bulls for characteristics of sperm plasma membrane, acrosome, and DNA integrity (P >  0.05). Relative abundances of PRM1 and PRM2 in sperm from the high and low fertility bulls were inversely related (P <  0.0001). Percentages of decondensed sperm were different between high and low fertility bulls (P < 0.0001) and total numbers of decondensed sperm were greater in low fertility bulls than high fertility bulls (R 2  = 0.72). Results of the present study are significant because molecular and morphological phenotypes of sperm that were detected affect fertility in livestock species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cryopreservation of semen and artificial insemination have an important, positive impact on cattle production, and product quality. Through the use of cryopreserved semen and artificial insemination, sperm from the best breeding bulls can be used to inseminate thousands of cows around the world. Although cryopreservation of bull sperm has advanced beyond that of other species, there are still major gaps in the knowledge and technology bases. Post-thaw viability of sperm is still low and differs significantly among the breeding bulls. These weaknesses are important because they are preventing advances both in fundamental science of mammalian gametes and reproductive biotechnology. Various extenders have been developed and supplemented with chemicals to reduce cryodamage or oxidative stress with varying levels of success. More detailed insights on sperm morphology and function have been uncovered through application of advanced tools in modern molecular and cell biology. This article provides a concise review of progress in the cryopreservation of bull sperm, advances in extender development, and frontiers using diverse techniques of the study of sperm viability. This scientific resource is important in animal biotechnology because with the advances in discovery of sperm fertility markers, there is an urgent need to improve post-thaw viability and fertility of sperm through enhanced cryopreservation for precision agriculture to produce food animals to ensure food security on the global scale.

Bull fertility, ability of the sperm to fertilize and activate the egg and support embryo development, is vital for cattle reproduction and production. Even though majority of histones are replaced by protamines, some histones are retained in sperm. It is known that chromatin remodeling during spermatogenesis results in dynamic changes in sperm chromatin structure through post-translational modifications (PTM) of sperm histones, which are important for regulation of gene expression. However, amounts of sperm Histone 4 (H4), its acetylated form (H4 acetyl), and to what extent these molecular attributes influence sperm chromatin structure and bull fertility are unknown. These gaps in the knowledge base are important because they are preventing advances in the fundamental science of bovine male gamete and improvement of bull fertility. The objective of this study was to test the hypothesis that expression dynamics as well as PTM of sperm H4 are associated with bull fertility. Flow cytometry was utilized to quantify H4 and H4 acetylated form in sperm from seven high and seven low fertility Holstein bulls. The results indicated that the average number of cells with H4 or H4 acetyl expression in high and low fertility bull sperm were 34.6 ± 20.4, 1.88 ± 1.8, 15.2 ± 20.8, and 1.4 ± 1.2, respectively. However, the sperm enriched in both H4 and H4 acetyl were different between high and low fertility groups (3.5 ± 0.6; 1.8 ± 0.8; P = 0.043). The localization and detection of H4 and H4 acetylation were measured by immunocytochemistry which revealed that H4 and H4 acetylation were equally distributed in the sperm head of high and low fertility sires. Western blotting results confirmed the presence of the H4 and its acetylated form in the sperm. Bioinformatics studies demonstrated that H4 is highly conserved among mammalians, and have significant gene ontology on spermatogenesis, early embryo implantation, and sperm capacitation. The results are significant because it demonstrates the replacement of canonical histone H4 into modified H4 acetylation in sperm and regulate its dynamics which is crucial for bull fertility and reproductive biotechnology. These findings advance fundamental science of mammalian early development and reproductive biotechnology.