Epigenetické změny spermií a jejich využití pro klinickou praxi v asistované reprodukci člověka

Základ zdravého embryonálního vývoje je položen už v průběhu gametogeneze. V současné době, v souladu s nárůstem procenta párů, které nemohu počít dítě přirozenou cestou, centra asistované reprodukce zaznamenávají svůj rozmach. Právě proto, porozumění biologie gamet a toho, co předávají embryu, je klíčem k léčbě neplodných párů a cestou k zdravému embryonálnímu vývoji. Proto se tato studie zaměřuje na epigenetický kód gamet, s prediktivním potenciálem, a na faktory, které epigenetický kód utváří. V souladu s předpokladem, že jsou vybrané molekulární faktory použitelné jako markéry kvality, byl studován vliv environmentálního polutantu bisfenolu S (BPS) na kvalitu zárodečných buněk a spermií s cílem identifikovat markery kvality gamet, použitelné pro screening spermií a jejich selekci pro účely asistované reprodukce. Pro dosáhnutí daných cílů byly použity vzorky lidských spermií, zárodečné buňky experimentálních myší kmene ICR a kančí spermie. Vzorky byly podrobeny analýzám pomocí průtokové cytometrie, immunocytochemie a western blotu. Experimentální práce byla schválena Etickou komisí FN Plzeň, resp. probíhala v souladu se schváleným projektem pokusu na experimentálních zvířatech. Studie provedená na lidských spermiích detekovala metylovaný histon H3 na lysinu K4 (H3K4me2) jako potencionální...Basement of healthy embryo development comes from quality of oocytes and spermatozoa. Today, when percentage of couples suffering infertility together with assisted reproductive therapy (ART) is increasing, understanding to gamete biology and heritable epigenetic code is crucial. The study is focused on promising epigenome based markers that could serve as indicators of gamete quality for either their screening or selection for ART. Accordingly selected markers were used for the investigation of environmental pollutant bisphenol S (BPS) effect on gametes quality. To obtain these aims, we have used human semen samples, boar semen samples and ICR mice gametes. Samples were analyzed by flow cytometry, immunocytochemistry and western blot analysis. All experimental work was in accordance with Ethics committee University Hospital in Pilsen and approved experimental designs for appropriate experimental animal project. In the study, we detected the dimethylation of histone H3 on lysine K4 (H3K4me2) as potential epigenetic marker of sperm quality and chromatin immaturity. Secondly, we observed the role of the gasotransmitter hydrogen sulphide (H2S) as anti-capacitating agents, slowing down capacitation possibly through post-translational modification of proteins. Thirdly, SIRT1 histone deacetylase was...Ústav histologie a embryologieLékařská fakulta v PlzniFaculty of Medicine in Pilse

Sperm selection by rheotaxis improves sperm quality and early embryo development

peer-reviewedThe objective of this work was to elucidate whether a sperm selection method that combines rheotaxis and microfluidics can improve the selection of spermatozoa over density gradient and swim-up. For this purpose human sperm selected by rheotaxis were compared against density gradient, swim-up and a control group of non-selected spermatozoa in split frozen-thawed (FT) and fresh (F) semen samples. Sperm quality was assessed in terms of motility, morphology, DNA fragmentation index (DFI), viability, acrosome integrity and membrane fluidity. Using a mouse model, we compared fertilisation and embryo development rates after performing ICSI with spermatozoa, sorted using rheotaxis or swim-up. Selection by rheotaxis yielded a sperm population with reduced DFI than the control (P < 0.05), improved normal morphology (P < 0.001) and higher total motility (TM; P < 0.001) than the other techniques studied in F and FT samples. Swim-up increased TM compared to density gradient and control in FT or F samples (P < 0.001), and yielded lower DFI than the control with F samples (P < 0.05). In FT samples, selection by rheotaxis yielded sperm with higher viability than control, density gradient and swim-up (P < 0.01) while acrosomal integrity and membrane fluidity were maintained. When mouse spermatozoa were selected for ICSI using rheotaxis compared to swim-up, there was an increase in fertilisation (P < 0.01), implantation (P < 0.001) and foetal development rates (P < 0.05). These results suggest that, in the absence of non-destructive DNA testing, the positive rheotaxis can be used to select a population of low DNA fragmentation spermatozoa with high motility, morphology and viability, leading to improved embryo developmental rates

Epignetic Modifications of the Sperm and the Application in Clinical Practice of Human Assisted Reproduction Therapy

Basement of healthy embryo development comes from quality of oocytes and spermatozoa. Today, when percentage of couples suffering infertility together with assisted reproductive therapy (ART) is increasing, understanding to gamete biology and heritable epigenetic code is crucial. The study is focused on promising epigenome based markers that could serve as indicators of gamete quality for either their screening or selection for ART. Accordingly selected markers were used for the investigation of environmental pollutant bisphenol S (BPS) effect on gametes quality. To obtain these aims, we have used human semen samples, boar semen samples and ICR mice gametes. Samples were analyzed by flow cytometry, immunocytochemistry and western blot analysis. All experimental work was in accordance with Ethics committee University Hospital in Pilsen and approved experimental designs for appropriate experimental animal project. In the study, we detected the dimethylation of histone H3 on lysine K4 (H3K4me2) as potential epigenetic marker of sperm quality and chromatin immaturity. Secondly, we observed the role of the gasotransmitter hydrogen sulphide (H2S) as anti-capacitating agents, slowing down capacitation possibly through post-translational modification of proteins. Thirdly, SIRT1 histone deacetylase was..

H3K4me2 accompanies chromatin immaturity in human spermatozoa: an epigenetic marker for sperm quality assessment

Research Communication. et al.Chromatin remodeling, including histone post-translational modifications, during spermatogenesis can affect sperm quality and fertility, and epigenetic marks may therefore be useful for clinical evaluations of sperm. Together with histone hyperacetylation, the dimethylation of histone H3 on lysine K4 (H3K4me2) is also required during protamination. Accordingly, we evaluated the utilization of this epigenetic mark for the identification of sperm with decrease quality and immature chromatin. In this study, 99 semen samples, including 22 normozoospermic (N), 63 asthenozoospermic (A), and 14 oligoasthenozoospermic (OA) samples, were comprehensively analyzed with respect to H3K4me2 levels, DNA damage (DNA fragmentation index, DFI), and sperm immaturity (high DNA stainability, %HDS), as determined by a sperm chromatin structure assay using flow cytometry. We detected a significant relationship between H3K4me2 and %HDS (r = 0.47; p A > OA) indicates the importance of chromatin immaturity and histone code deviations in sperm evaluations. Using various approaches, our study elucidated H3K4me2 as a molecular marker of sperm quality with potential use in reproductive medicine.This work was supported by the University of Castilla-La Mancha and Junta de Comunidades de Castilla-La Mancha [SBPLY/17/180501/000470]; National Sustainability Programme I provided by the Ministry of Education, Youth and Sports of the Czech Republic [Nr. LO1503]; Ministry of Education, Youth and Sports of the Czech Republic [No. SVV 02690]; Charles University Research Fund [PROGRES Q39]; Ministry of Education, Youth and Sports of the Czech Republic and financed from The European Regional Development Fund [No. CZ.02.1.01/0.0/0.0/16_019/0000787 “Fighting]; University of Missouri [F21C program].Peer reviewe

Genome-wide association study reveals candidate markers related to field fertility and semen quality traits in Holstein-Friesian bulls

In vitro assessment of bull semen quality is routinely used in bull semen processing centres in order to ensure that semen destined to be used in the field has passed minimum standards. Despite these stringent quality control checks, individual bulls that pass the quality control checks can still vary in field fertility by up to 25%. A genome-wide association study was undertaken to determine genetic markers associated with prefreeze and post-thaw bull sperm quality traits as well as field fertility. Genome-wide association analysis was performed using a single nucleotide polymorphism (SNP) regression mixed linear model in WOMBAT. Genes within a 250 Kb span of a suggestive (P ≤ 1 × 10−5) SNP were considered as candidate genes. One SNP was associated with adjusted pregnancy rate, and 21 SNPs were associated across the seven semen quality traits (P ≤ 1 × 10−5). Functional candidate genes include SIPA1L2 which was associated with adjusted pregnancy rate. This encodes a Rap GTPase-activating protein involved in Rap1 signalling pathway and was previously found to play a role in the process of sperm differentiation. Gene ontology (GO) analysis also identified significantly enriched biological processes involved protein tyrosine kinase activity including genes such as DYRK1A, TEC and TXK that were associated with sperm motility prior to freezing. Another candidate gene associated with post-thaw sperm motility was FHDC1 which coordinates actin filament and microtubule dynamics. The induced 11 GO terms in the ejaculates rejected after freezing trait were related to ATPase, phosphatase and hydrolase activity. These results reveal novel specific genomic regions and candidate genes associated with economically important phenotypes such as field fertility and semen quality traits

Genome-wide association study reveals candidate markers related to field fertility and semen quality traits in Holstein-Friesian bulls

 In vitro assessment of bull semen quality is routinely used in bull semen processing centres in order to ensure that semen destined to be used in the field has passed minimum standards. Despite these stringent quality control checks, individual bulls that pass the quality control checks can still vary in field fertility by up to 25%. A genome-wide association study was undertaken to determine genetic markers associated with prefreeze and post-thaw bull sperm quality traits as well as field fertility. Genome-wide association analysis was performed using a single nucleotide polymorphism (SNP) regression mixed linear model in WOMBAT. Genes within a 250 Kb span of a suggestive (P ≤ 1 × 10−5) SNP were considered as candidate genes. One SNP was associated with adjusted pregnancy rate, and 21 SNPs were associated across the seven semen quality traits (P ≤ 1 × 10−5). Functional candidate genes include SIPA1L2 which was associated with adjusted pregnancy rate. This encodes a Rap GTPase-activating protein involved in Rap1 signalling pathway and was previously found to play a role in the process of sperm differentiation. Gene ontology (GO) analysis also identified significantly enriched biological processes involved protein tyrosine kinase activity including genes such as DYRK1A, TEC and TXK that were associated with sperm motility prior to freezing. Another candidate gene associated with post-thaw sperm motility was FHDC1 which coordinates actin filament and microtubule dynamics. The induced 11 GO terms in the ejaculates rejected after freezing trait were related to ATPase, phosphatase and hydrolase activity. These results reveal novel specific genomic regions and candidate genes associated with economically important phenotypes such as field fertility and semen quality traits. </p

Sperm DNA methylation patterns at discrete CpGs and genes involved in embryonic development are related to bull fertility

Background: Despite a multifactorial approach being taken for the evaluation of bull semen quality in many animal breeding centres worldwide, reliable prediction of bull fertility is still a challenge. Recently, attention has turned to molecular mechanisms, which could uncover potential biomarkers of fertility. One of these mechanisms is DNA methylation, which together with other epigenetic mechanisms is essential for the fertilising sperm to drive normal embryo development and establish a viable pregnancy. In this study, we hypothesised that bull sperm DNA methylation patterns are related to bull fertility. We therefore investigated DNA methylation patterns from bulls used in artifcial insemination with contrasting fertility scores. Results: The DNA methylation patterns were obtained by reduced representative bisulphite sequencing from 10 high-fertility bulls and 10 low-fertility bulls, having average fertility scores of −6.6 and + 6.5%, respectively (mean of the population was zero). Hierarchical clustering analysis did not distinguish bulls based on fertility but did highlight individual diferences. Despite this, using stringent criteria (DNA methylation diference≥35% and a q-value Conclusion: This study demonstrated that at specifc CpG sites, sperm DNA methylation status is related to bull fertility, and identifed seven diferently methylated genes in sperm of subfertile bulls that may lead to altered gene expression and potentially infuence embryo development.</p

Adenylate kinase 9 is essential for sperm function and male fertility in mammals

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.ISSN:0027-8424ISSN:1091-649