Effects of photobiomodulation therapy on human sperm function / Efectos de la terapia de fotobiomodulación en la función del esperma humano

Introduction: Sperm motility is a crucial factor in male infertility and it depends on mitochondrial tail movements. Photobiomodulation light therapy allows the cells to produce their energy through activation of the mitochondria. The aim of the present study was to examine the impact of photobiomodulation on sperm motility in astenozoospermic individuals. Materials and methods: Following semen analyses of 20 astenozoospermic individuals, collected semen samples were centrifuged. Pellet was obtained and homogenized through mixing with culture media in 1:1 ratio. Each semen samples were divided into 3 groups. In the first group, control samples were not exposed to laser irradiation. The Group 2 and Group 3 were exposed to 650nm wavelength of photobiomodulation from 10cm distance in dark environment via a 36cm2 aperture sizer with 200mW output power for 30 and 60min duration, respectively. Sperm motilities were evaluated and chromatin condensation of sperms was determined. Results: Sperm motilities were significantly increased in photobiomodulation groups compared with the controls. Sperm motilities tended to be different between the 30 and 60min red light exposure groups; however, it was not statistically significant. When the motility grades were compared, no significant difference was observed in non-progressive motility sperms. While immotile sperms decreased significantly in the photobiomodulation groups compared to the control group, progressive sperms increased. (AU)

Introducción: La motilidad espermática es un factor crucial en la infertilidad masculina y depende de los movimientos de la cola mitocondrial. La fototerapia de fotobiomodulación permite que las células produzcan su energía a través de la activación de las mitocondrias. El objetivo del presente estudio fue examinar el impacto de la fotobiomodulación en la motilidad de los espermatozoides en individuos astenozoospérmicos. Materiales y métodos: Luego de los análisis de semen de 20 individuos astenozoospérmicos, se centrifugaron las muestras de semen recolectadas. Se obtuvo el sedimento y se homogeneizó mezclándolo con medios de cultivo en una proporción de 1:1. Cada muestra de semen se dividió en 3 grupos. En el primer grupo, las muestras de control no se expusieron a la irradiación láser. El grupo 2 y el grupo 3 fueron expuestos a una longitud de onda de 650nm de fotobiomodulación desde una distancia de 10cm en un ambiente oscuro a través de un medidor de apertura de 36cm2 con una potencia de salida de 200mW durante 30 y 60min de duración, respectivamente. Se evaluaron las motilidades de los espermatozoides y se determinó el tamaño de la cromatina de los espermatozoides. Resultados: La motilidad de los espermatozoides aumentó significativamente en los grupos de fotobiomodulación en comparación con los controles. La motilidad de los espermatozoides tendió a ser diferente entre los grupos de exposición a la luz roja de 30 y 60min; sin embargo, no fue estadísticamente significativo. Cuando se compararon los grados de motilidad, no se observaron diferencias significativas en los espermatozoides de motilidad no progresiva. Mientras que los espermatozoides inmóviles disminuyeron significativamente en los grupos de fotobiomodulación en comparación con el grupo de control, los espermatozoides progresivos aumentaron. (AU)

iTRAQ-based comparative proteomics reveal an enhancing role of PRDX6 in the freezability of Mediterranean buffalo sperm.

BACKGROUND: Semen cryopreservation is a critical tool for breed improvement and preservation of biodiversity. However, instability of sperm freezability affects its application. The Mediterranean buffalo is one of the river-type buffaloes with the capacity for high milk production. Until now, there is no specific cryopreservation system for Mediterranean buffalo, which influences the promotion of excellent cultivars. To improve the semen freezing extender used in cryopreservation of Mediterranean buffalo, different protein datasets relating to freezability sperm were analyzed by iTRAQ-based proteomics. This study will be beneficial for further understanding the sperm freezability mechanism and developing new cryopreservation strategy for buffalo semen. RESULTS: 2652 quantified proteins were identified, including 248 significantly differentially expressed proteins (DEP). Gene Ontology (GO) analysis indicated that many these were mitochondrial proteins, enriched in the molecular function of phospholipase A2 activity and enzyme binding, and biological processes of regulation of protein kinase A signaling and motile cilium assembly. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 17 significant pathways, including oxidative phosphorylation (OXPHOS). Furthermore, 7 DEPs were verified using parallel reaction monitoring or western blot, which confirmed the accuracy of the iTRAQ data. Peroxiredoxin 6 (PRDX6), which expressed 1.72-fold higher in good freezability ejaculate (GFE) compared to poor freezability ejaculate (PFE) sperms, was selected to explore the function in sperm freezability by adding recombinant PRDX6 protein into the semen freezing extender. The results showed that the motility, mitochondrial function and in vitro fertilization capacity of frozen-thawed sperm were significantly increased, while the oxidation level was significantly decreased when 0.1 mg/L PRDX6 was added compared with blank control. CONCLUSIONS: Above results revealed the metabolic pattern of freezability of Mediterranean buffalo sperms was negatively associated with OXPHOS, and PRDX6 had protective effect on cryo-damage of frozen-thawed sperms.

Intergenerational response to sperm competition risk in an invasive mammal.

Studies of socially mediated phenotypic plasticity have demonstrated adaptive male responses to the 'competitive' environment. Despite this, whether variation in the paternal social environment also influences offspring reproductive potential in an intergenerational context has not yet been examined. Here, we studied the descendants of wild-caught house mice, a destructive pest species worldwide, to address this knowledge gap. We analysed traits that define a 'competitive' phenotype in the sons of males (sires) that had been exposed to either a high-male density (competitive) or high-female density (non-competitive) environment. We report disparate reproductive strategies among the sires: high-male density led to a phenotype geared for competition, while high-female density led to a phenotype that would facilitate elevated mating frequency. Moreover, we found that the competitive responses of sires persisted in the subsequent generation, with the sons of males reared under competition having elevated sperm quality. As all sons were reared under common-garden conditions, variation in their reproductive phenotypes could only have arisen via nongenetic inheritance. We discuss our results in relation to the adaptive advantage of preparing sons for sperm competition and suggest that intergenerational plasticity is a previously unconsidered aspect in invasive mammal fertility control.

Polymorphic Rearrangements of Human Chromosome 9 and Male Infertility: New Evidence and Impact on Spermatogenesis.

Chromosomal polymorphisms are structural variations in chromosomes that define the genomic variance of a species. These alterations are recurrent in the general population, and some of them appear to be more recurrent in the infertile population. Human chromosome 9 is highly heteromorphic, and how its rearrangement affects male fertility remains to be fully investigated. In this study, we aimed to investigate the association between the polymorphic rearrangements of chromosome 9 and male infertility via an Italian cohort of male infertile patients. Cytogenetic analysis was carried out, along with Y microdeletion screening, semen analysis, fluorescence in situ hybridization, and TUNEL assays using spermatic cells. Chromosome 9 rearrangements were observed in six patients: three of them showed a pericentric inversion, while the others showed a polymorphic heterochromatin variant 9qh. Of these, four patients exhibited oligozoospermia associated with teratozoospermia, along with a percentage of aneuploidy in the sperm of above 9%, in particular, an increase in XY disomy. Additionally, high values for sperm DNA fragmentation (≥30%) were observed in two patients. None of them had microdeletions to the AZF loci on chromosome Y. Our results suggest that polymorphic rearrangements of chromosome 9 might be associated with abnormalities in sperm quality due to incorrect spermatogenesis regulation.

Antifreeze protein type III addition to freezing extender comprehensively improves post-thaw sperm properties in Okinawan native Agu pig.

Sperm cryopreservation often leads to physical cell damage through ice crystal formation. This study evaluates the improvements to freezing extender cryoprotective activity due to antifreeze protein (AFP) addition, which primarily acts on ice crystal formation, through investigating the post-thaw sperm properties of Okinawan native Agu pig. Six individual boar sperm samples were diluted with the freezing extender supplemented with 1 µg/mL of AFP I or AFP III and then subjected to cryopreservation. Treatment with AFP I during the freezing procedure had no improvement for any characteristics after thawing compared to untreated sperm. In contrast, the addition of AFP III to the freezing extender strongly increased sperm motility, mitochondria and cell membrane integrity, and the acrosomal proteolytic activity of frozen-thawed sperm in 5 of 6 individuals (P < 0.05). Furthermore, cryoinjury prevention by AFP III significantly enhanced sperm viability (by ATP content), and maintained DNA quality and in vitro sperm penetrability compared with AFP I treatment (P < 0.05). These findings demonstrate that AFP III addition to the freezing extender of boar sperm is more effective in maintaining sperm characteristics than the extender without AFP III or AFP I, despite individual differences in response.

Ram sperm cryopreservation disrupts metabolism of unsaturated fatty acids.

In ram sperm, metabolites are important components of the plasma membrane, energy metabolism cycle, and precursors for other membrane lipids, and they may have important roles in maintaining plasma membrane integrity, energy metabolism, and regulation of cryotolerance. In this study, the ejaculates from 6 Dorper rams were pooled and sperm were systematically investigated by metabolomics at various steps of cryopreservation (37 °C, fresh [F]; from 37 to 4 °C, cooling [C]; and from 4 to -196 to 37 °C, frozen-thawed [FT]) to identify differential metabolites (DM). There were 310 metabolites identified, of which 86 were considered DMs. Regarding the DMs, there were 23 (0 up and 23 down), 25 (12 up and 13 down), and 38 (7 up and 31 down) identified during cooling (C vs F), freezing (FT vs C), and cryopreservation (FT vs F), respectively. Furthermore, some key polyunsaturated fatty acids (FAs), particularly, linoleic acid (LA), docosahexaenoic acid (DHA), and arachidonic acid (AA) were down-regulated during cooling and cryopreservation. Significant DMs were enriched in several metabolic pathways including biosynthesis of unsaturated FAs, LA metabolism, mammalian target of rapamycin (mTOR), forkhead box transcription factors (FoxO), adenosine monophosphate-activated protein kinase (AMPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathways, regulation of lipolysis in adipocytes, and FA biosynthesis. This was apparently the first report to compare metabolomics profiles of ram sperm during cryopreservation and provided new knowledge to improve this process.

A new role of H89: Reduces capacitation-like changes through inhibition of cholesterol efflux, calcium influx, and proteins tyrosine phosphorylation during sperm cryopreservation in buffalo.

It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 µM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 µM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process.

Time of day modified the time required for semen collection with electroejaculation and slightly affected the quality of fresh semen in rams.

The aim was to compare some stress responses to electroejaculation (EE), and the quality of fresh semen, when ram semen is collected at dawn (06:00 h), noon (12:00 h), or evening (18:00 h). Twelve Corriedale rams were used, and semen was collected from four rams at each study time on three different days, with a Latin-square design. The time required for EE, the number of vocalizations emitted, heart rate, and rectal temperature were recorded, and fresh semen was evaluated. The time required for EE was shorter at evening than at dawn and noon (399.3 s, 480.6 s, and 460.2 s respectively; pooled SEM = 72.1; P = 0.03). The percentage of sperm with progressive motility was greater at noon than dawn (59.7% and 50.3%; pooled SEM = 5.8; P = 0.05). Curvilinear velocity was greater at dawn than evening (117.0 µm/s and 95.5 µm/s; pooled SEM = 7.1; P = 0.04), slow linear velocity was greater at evening than at dawn and noon (13.1 µm/s, 9.3 µm/s, and 8.5 µm/s respectively; pooled SEM = 1.7, P = 0.05), and the slow average path velocity was greater at evening than dawn and noon (16.2 µm/s, 11.7 µm/s, and 10.8 µm/s respectively; pooled SEM = 1.9, P = 0.05). In conclusion, the collection time modified the time required for electroejaculation and had only slight effects on the quality of fresh semen. Overall, the time of the day appears to have only slight effects on semen collection and quality.

Increased male investment in sperm competition results in reduced maintenance of gametes.

Male animals often show higher mutation rates than their female conspecifics. A hypothesis for this male bias is that competition over fertilization of female gametes leads to increased male investment into reproduction at the expense of maintenance and repair, resulting in a trade-off between male success in sperm competition and offspring quality. Here, we provide evidence for this hypothesis by harnessing the power of experimental evolution to study effects of sexual selection on the male germline in the seed beetle Callosobruchus maculatus. We first show that 50 generations of evolution under strong sexual selection, coupled with experimental removal of natural selection, resulted in males that are more successful in sperm competition. We then show that these males produce progeny of lower quality if engaging in sociosexual interactions prior to being challenged to surveil and repair experimentally induced damage in their germline and that the presence of male competitors alone can be enough to elicit this response. We identify 18 candidate genes that showed differential expression in response to the induced germline damage, with several of these previously implicated in processes associated with DNA repair and cellular maintenance. These genes also showed significant expression changes across sociosexual treatments of fathers and predicted the reduction in quality of their offspring, with expression of one gene also being strongly correlated to male sperm competition success. Sex differences in expression of the same 18 genes indicate a substantially higher female investment in germline maintenance. While more work is needed to detail the exact molecular underpinnings of our results, our findings provide rare experimental evidence for a trade-off between male success in sperm competition and germline maintenance. This suggests that sex differences in the relative strengths of sexual and natural selection are causally linked to male mutation bias. The tenet advocated here, that the allocation decisions of an individual can affect plasticity of its germline and the resulting genetic quality of subsequent generations, has several interesting implications for mate choice processes.

Alternative signal pathways underly fertilization and egg activation in a fish with contrasting modes of spawning.

BACKGROUND: The processes of fertilization and egg activation are vital for early embryogenesis. However, while the mechanisms associated with key events during these processes differ among species and modes of spawning, the signal pathways underlying these processes are opaque for many fishes, including economically important species. RESULTS: We investigated phenotypic traits, ultrastructure and protein expression levels in the eggs of the topmouth culter (Culter alburnus), a protected and economically important freshwater fish that exhibits two spawning modes, producing semi-buoyant eggs and adhesive eggs. Unfertilized eggs of C. alburnus were examined, as well as eggs at fertilization and 30 min post fertilization. Our results showed that in semi-buoyant eggs, energy metabolism was activated at fertilization, followed by elevated protein expression of cytoskeleton and extracellular matrix (ECM)-receptor interactions that resulted in rapid egg swelling; a recognized adaptation for lotic habitats. In contrast, in adhesive eggs fertilization initiated the process of sperm-egg fusion and blocking of polyspermy, followed by enhanced protein expression of lipid metabolism and the formation of egg envelope adhesion and hardening, which are adaptive in lentic habitats. CONCLUSION: Our findings indicate that alternative signal pathways differ between modes of spawning and timing during the key processes of fertilization and egg activation, providing new insights into the molecular mechanisms involved in adaptive early embryonic development in teleost fishes.

Proteomic analysis reveals the potential positive effects of Mito-TEMPO on ram sperm motility and fertility during cryopreservation.

The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 µM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT40 groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 µM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT40 group. The inclusion of 40 µM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.

Antioxidant additive melatonin in tris-based egg yolk extender improves post-thaw sperm attributes in Hariana bull.

In the study, melatonin, a known antioxidant pineal peptide was used as an additive in the tris-egg yolk glycerol-based semen extender in Hariana bull semen and post-thaw sperm characters were evaluated. In the study, Group I was a control without melatonin; and Group II, III, and IV were having 0.5 mM, 1 mM, and 2 mM melatonin/80 × 106 spermatozoa, respectively were treatment groups. Thirty-two semen ejaculates from 4 Hariana bulls were processed for freezing and post-thaw sperm characteristics were evaluated. Sperm motility, velocity, the viability with intact membrane, and total antioxidant capacity were markedly (P < 0.05) improved in Group IV compared to all other groups. The lipid peroxidation and total protein carbonylation were substantially (P < 0.05) decreased in Group IV compared to all other groups. The population of cryocapacitated, acrosome-reacted, and apoptotic-like spermatozoa were significantly (P < 0.05) decreased in Group IV. Further, the relative band intensity of 74 kDa protein and percent of spermatozoa showing positive immune reactivity to tyrosine-phosphorylated proteins was decreased in Group IV. The progesterone-receptor ligand binding, in vitro capacitation response, and Vanguard distance were markedly (P < 0.05) improved in Group IV. In summary- Group IV having 2 mM melatonin was found to be optimal in providing cryoprotective effects to Hariana bull spermatozoa after freezing-thawing and can be suitably used as a semen additive during semen cryopreservation.

Paradigm shift in frog sperm cryopreservation: reduced role for non-penetrating cryoprotectants.

In brief: Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs. Abstract: Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.

A review of the use of antioxidants in bovine sperm preparation protocols.

Reactive oxygen species (ROS) and oxidative stress (OS), the imbalance between the production of free radicals and the cellular antioxidant defenses, are discussed in relation to their role in bovine sperm physiology. Oxidative stress has been associated to male infertility and low fertility rates in Assisted Reproductive Techniques (ART). Antioxidant supplementation is an interesting approach to overcome OS-related infertility and assisted reproduction drawbacks. Several studies have been conducted to identify the potential sources of ROS in a typical ART setting and the impact of antioxidant supplementation on semen quality and pregnancy outcome. Procedures such as freezing and thawing, centrifugation and incubation are thought to produce significant amounts of ROS with a negative impact on sperm quality parameters and reproductive competence. Given the important role of ROS in sperm function, the addition of antioxidants in sperm media to prevent OS and to improve the reproductive outcome requires attention. Currently, there is limited evidence to support the ameliorative effect of antioxidant supplementation on fertilization and embryo development in farm animals. This review summarizes the different types and concentrations of antioxidants used in sperm preparation media of bovine species and their effectiveness in neutralizing excessive ROS production while preserving physiological sperm function.

In vitro exposure to environmentally relevant concentrations of norgestrel affects sperm physiology and reproductive success of the Pacific oyster Crassostrea gigas.

Progestins in aquatic environments are of increasing concern, as shown by the results of toxicological studies on adult invertebrates with external fertilization. However, their potential effects on the gametes and reproductive success of such animals remain largely unknown. Thus, the current study assessed the effect of in vitro exposure of environmentally relevant concentrations (10 ng/L and 1000 ng/L) of norgestrel (NGT) on the sperm of Pacific oyster Crassostrea gigas, analyzing sperm motility, ultrastructure, mitochondrial function, ATP status, characteristic enzyme activities, and DNA integrity underlying fertilization and hatching success. The results showed that NGT increased the percentage of motile sperm by elevating intracellular Ca2+ levels, Ca2+-ATPase activity, creatine kinase activity, and ATP content. Although superoxide dismutase activity was enhanced to eliminate reactive oxygen species generated by NGT, oxidative stress occurred, as indicated by the increase in malonaldehyde content and damage to plasma membranes and DNA. As a consequence, fertilization rates decreased. However, hatching rates did not alter significantly, possibly as a result of DNA repair processes. This study demonstrates oyster sperm as a useful, sensitive tool for toxicological research of progestins and provides ecologically relevant information on reproductive disturbance in oysters resulting from exposure to NGT.

Characteristics of oocytes that failed to fertilize after intracytoplasmic sperm injection.

Background: We assessed the morphologies of meiotic spindles in oocytes that failed to fertilize following intracytoplasmic sperm injection (ICSI) and identified factors contributing to failed fertilization. Methods: A total of 225 unfertilized oocytes were collected after ICSI. Oocytes were fixed and stained for tubulin and chromosomes. Meiotic spindle morphologies, chromosome alignment, and sperm nuclear decondensation were assessed to identify contributing factors to fertilization failure. We identified relationships between several factors and both abnormal spindle morphologies and sperm nuclear decondensation in oocytes that failed to fertilize. Results: Three causes for unfertilized oocytes after ICSI were identified: (I) the absence of a sperm nucleus in the ooplasm; (II) failed oocyte activation; and (III) defects in pronucleus formation or migration. The rate of disarranged polar spindles in oocytes collected from women older than 35 years (73.3%; 33/45 oocytes) was significantly higher than that of those collected from women 35 years and younger (50.4%; 68/135 oocytes; odds ratio [OR]: 2.71, 95% confidence interval [CI]: 1.29-5.69, p = 0.009). The proportion of unfertilized oocytes with abnormal spindles and chromosome misalignment was significantly higher in oocytes collected from women older than 35 years than those from women 35 years and younger (62.2% vs. 41.5%, p = 0.016). The proportion of partially decondensed chromatin in the abnormal sperm morphology group was significantly higher than in the normal sperm morphology group (66.7% versus 52.9%, OR: 1.78, 95% CI: 1.01-3.11, p = 0.044). Conclusions: The main contributor to the failure of oocytes to fertilize after ICSI is failed oocyte activation. The ICSI technique used, the maternal age, and sperm morphology are also contributing factors in fertilization failure after ICSI.

Sperm physiology and in vitro fertilising ability rely on basal metabolic activity: insights from the pig model.

Whether basal metabolic activity in sperm has any influence on their fertilising capacity has not been explored. Using the pig as a model, the present study investigated the relationship of energetic metabolism with sperm quality and function (assessed through computer-assisted sperm analysis and flow cytometry), and fertility (in vitro fertilisation (IVF) outcomes). In semen samples from 16 boars, levels of metabolites related to glycolysis, ketogenesis and Krebs cycle were determined through a targeted metabolomics approach using liquid chromatography-tandem mass spectrometry. High-quality sperm are associated to greater levels of glycolysis-derived metabolites, and oocyte fertilisation and embryo development are conditioned by the sperm metabolic status. Interestingly, glycolysis appears to be the preferred catabolic pathway of the sperm giving rise to greater percentages of embryos at day 6. In conclusion, this study shows that the basal metabolic activity of sperm influences their function, even beyond fertilisation.

Machine learning-based clustering to identify the combined effect of the DNA fragmentation index and conventional semen parameters on in vitro fertilization outcomes.

BACKGROUND: Previous studies have demonstrated an association between male sperm quality and assisted reproduction outcomes, focusing on the effects of individual parameters and reaching controversial conclusions. The WHO 6th edition manual highlights a new semen assay, the sperm DNA fragmentation index, for use after routine semen examination. However, the combined effect of the sperm DNA fragmentation index (DFI) and routine semen parameters remains largely unknown. METHODS: We assessed the combined effect of the sperm DFI and conventional semen parameters on single fresh conventional IVF outcomes for infertile couples from January 1, 2017, to December 31, 2020. IVF outcomes were obtained from the cohort database follow-up records of the Clinical Reproductive Medicine Management System of the Third Affiliated Hospital of Guangzhou Medical University. An unsupervised K-means clustering method was applied to classify participants into several coexposure pattern groups. A multivariate logistic regression model was used for statistical analysis. RESULTS: A total of 549 live births among 1258 couples occurred during the follow-up period. A linear exposure-response relationship was observed among the sperm DFI, sperm motility, and IVF outcomes. In multivariable adjustment, increased sperm DFI values and decreased sperm motility and semen concentration levels were associated with reduced odds of favourable IVF outcomes. Four coexposure patterns were generated based on the sperm DFI and the studied semen parameters, as follows: Cluster 1 (low sperm DFI values and high sperm motility and semen concentration levels), Cluster 2 (low sperm DFI values and moderate sperm motility and semen concentration levels), Cluster 3 (low sperm DFI values and low sperm motility and semen concentration levels) and Cluster 4 (high sperm DFI values and low sperm motility and semen concentration levels). Compared with those in Cluster 1, participants in Cluster 3 and Cluster 4 had lower odds of a live birth outcome, with odds ratios (95% confidence intervals [CIs]) of 0.733 (0.537, 0.998) and 0.620 (0.394, 0.967), respectively. CONCLUSIONS: When combined with low sperm DFI values, there was no significant difference between high or moderate sperm concentration and motility levels, and both were associated with favourable IVF outcomes. Low sperm parameter levels, even when DFI values remain low, may still lead to poor IVF outcomes. Participants with high sperm DFI values and low sperm motility and semen concentration levels had the worst outcomes. Our findings offer a novel perspective for exploring the joint effects of sperm DFI and routine semen parameter values.

Effects of therapeutic ultrasound and moderate heat on stallion testes.

Transplantation of stem cells into dysfunctional testes is currently being investigated as a therapeutic option for men and stallions with advanced testicular degeneration. This series of "proof of concept" studies aimed to identify a safe and efficient method of inducing severe testicular degeneration to create an optimal equine recipient model for intratesticular stem cell transplantation (SCT). Two ex vivo and two in vivo experiments were conducted. At first, forty testes obtained from castrations were used to identify an effective therapeutic ultrasound (TUS) device and the protocol for increasing intratesticular temperature in stallions. Six min of treatment using the Vetrison Clinic Portable TUS machine raised the intratesticular temperature by 8°C-12.5 °C. This protocol was applied to treat three scrotal testes in three Miniature horse stallions, three times, every other day. Contralateral testes served as controls. There were signs of slight tubular degeneration in treated testes two and three weeks after TUS treatment. The number of seminiferous tubules (STs) with exfoliated germ cells (GCs) was increased in one testis only, three weeks after treatment. The degree of apoptosis of GCs was higher in each treated testis in comparison to the contralateral control testis. Next, the ability of various heating devices to increase intratesticular temperatures to at least 43 °C in stallion testes was tested, using twenty testes obtained from castrations. ThermaCare® Lower Back & Hip Pain Therapy Heatwrap (TC heat wrap) reliably increased intratesticular temperatures and kept them continuously between 43 °C and 48 °C for seven to 8 h. In the follow-up in vivo study, the left testes of three Miniature horse stallions were treated with TUS, after which both testes of each stallion were treated with moderate heat provided by the TC heat wrap (three times, every other day, for 5 h each time). There were signs of moderate tubular degeneration in the samples from all treated testes obtained three weeks after treatments (Heat only or Heat/TUS): areas with hypospermatogenesis, spermatogenic arrest, vacuolized Sertoli cells, numerous STs with exfoliated GCs, increased degree of GCs apoptosis, and changes in three histomorphometric numeric attributes of STs. We concluded that TUS or TC wraps increase intratesticular temperature of the isolated stallion testes. Further, treatment with TUS or moderate heat may induce mild to moderate degenerative changes in stallion testes. However, to achieve more robust result - severe testicular degeneration, our treatment protocol has to be modified.

Cryopreservation modifies the distribution of the prostate-derived lectin SL15 on the llama (Lama glama) sperm.

Lectin-like molecules play a key role in mammalian sperm functionality. These multifunctional proteins have been proven to be involved in sperm capacitation, sperm motility, and viability, formation of the oviductal sperm reservoir, and in sperm-oocyte interaction. In a previous study, we reported the presence of a novel seminal plasma lectin, sperm lectin 15 kDa (SL15), adsorbed to the llama sperm. In order to gain knowledge in the understanding of SL15 and its functions, the aims of this study were to (a) elucidate the presence and localization of SL15 in the llama male reproductive tract and sperm, and (b) determine whether the sperm cryopreservation process of cooling and freeze-thawing affects the SL15 levels and distribution on llama sperm. We found that SL15 protein was expressed along the male reproductive system: testis, epididymis, prostate, and bulbourethral glands, being the prostate the main site of SL15 secretion. SL15 was localized on the sperm head, following different localization patterns. In order to understand if sperm cryopreservation induces modifications in the SL15 adsorption pattern, immunocytochemistry and flow cytometry analysis were carried out on fresh, 24 h cooled, and frozen-thawed sperm. Both cooled and frozen sperm showed particular SL15 patterns, that were not observed in the freshly ejaculated, indicating loss of SL15. Flow cytometry analysis also exhibited a decrease of SL15 in the cooled sperm (P < 0.05), whereas a tendency to decrease was found in frozen-thawed sperm (P < 0.1) when compared with freshly ejaculated sperm. This study extends the knowledge about the SL15 in the llama male physiology and provides evidence that cryopreservation-related techniques disrupt SL15 adsorption to the sperm membrane, possibly affecting sperm functionality and fertility.