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Methylation analyses of GNAS XL DMR of BSAS of two normal controls (top a and b) and two semen samples from asthenospermic group (c) and oligoasthenospermic group (d). Mean methylation levels and number of reads are given below each pattern. Lines represent reads; columns represent CpG dinucleotides; blue squares represent unmethylated CpGs; red squares represent methylated CpGs; white squares represent missing sequence information
Source publication
PurposeChanges in DNA methylation modifications have been associated with male infertility. With the development of assisted reproductive technologies (ARTs), abnormal DNA methylation in sperm, especially in imprinted genes, may impact the health of offspring and requires an in-depth study.Methods
In this study, we collected abnormal human semen sa...
Citations
... Of note, the identified DMRs exhibited a prevalent hypermethylation pattern [19]. The present study also revealed that these hypermethylated DMRs were primarily located within gene bodies, in line with earlier research on male infertility and sperm methylation [37][38][39]. Taken together, these results indicated that the DNA methylation pattern of spermatozoa may be distinct compared with that of somatic cells, challenging the conventional belief that methylation is principally enriched in the promoter region. ...
Background
Spermatogenesis is a highly regulated and complex process in which DNA methylation plays a crucial role. This study aimed to explore the differential methylation profiles in sperm DNA between patients with asthenospermia (AS) and healthy controls (HCs), those with oligoasthenospermia (OAS) and HCs, and patients with AS and those with OAS.
Results
Semen samples and clinical data were collected from five patients with AS, five patients with OAS, and six age-matched HCs. Reduced representation bisulfite sequencing (RRBS) was performed to identify differentially methylated regions (DMRs) in sperm cells among the different types of patients and HCs. A total of 6520, 28,019, and 16,432 DMRs were detected between AS and HC, OAS and HC, and AS and OAS groups, respectively. These DMRs were predominantly located within gene bodies and mapped to 2868, 9296, and 9090 genes in the respective groups. Of note, 12, 9, and 8 DMRs in each group were closely associated with spermatogenesis and male infertility. Furthermore, BDNF, SMARCB1, PIK3CA, and DDX27; RBMX and SPATA17; ASZ1, CDH1, and CHDH were identified as strong differentially methylated candidate genes in each group, respectively. Meanwhile, the GO analysis of DMR-associated genes in the AS vs. HC groups revealed that protein binding, cytoplasm, and transcription (DNA-templated) were the most enriched terms in the biological process (BP), cellular component (CC), and molecular function (MF), respectively. Likewise, in both the OAS vs. HC and AS vs. OAS groups, GO analysis revealed protein binding, nucleus, and transcription (DNA-templated) as the most enriched terms in BP, CC, and MF, respectively. Finally, the KEGG analysis of DMR-annotated genes and these genes at promoters suggested that metabolic pathways were the most significantly associated across all three groups.
Conclusions
The current study results revealed distinctive sperm DNA methylation patterns in the AS vs. HC and OAS vs. HC groups, particularly between patients with AS and those with OAS. The identification of key genes associated with spermatogenesis and male infertility in addition to the differentially enriched metabolic pathways may contribute to uncovering the potential pathogenesis in different types of abnormal sperm parameters.
... By next-generation bisulfite sequencing, another study demonstrated that differentially methylated regions (DMRs) of imprinted genes, including SNRPN, MEG8, GNAS, and H19 revealed distinct methylation patterns in the abnormal semen groups [18]. ...
Male infertility accounts for nearly 40%–50% of all infertile cases. One of the most prevalent disorders detected in infertile men is errors in the MEST differentially methylated region (DMR), which has been correlated with poor sperm indexes. The aim of our study was to characterize the methylation pattern of the MEST gene, along with assessing seminal factors and chromatin condensation in sperm samples from both infertile patients and fertile cases, all of whom were candidates for intracytoplasmic sperm injection. We collected forty-five semen specimens from men undergoing routine spermiogram analysis at the Infertility Treatment Center. The specimens consisted of 15 samples of normospermia as the control group, 15 individuals of asthenospermia, and 15 individuals of oligoasthenoteratospermia as the cases group. Standard semen analysis and the chromatin quality and sperm maturity tests using aniline blue staining were performed. The DNA from spermatozoa was extracted and treated with a sodium bisulfite–based procedure. The methylation measure was done quantitatively at the DMRs of the MEST gene by quantitative methylation-specific polymerase chain reaction (qMSP). The mean percentages of total motility, progression, and morphology in normospermia were significantly higher than oligoasthenoteratospermia and asthenospermia, and they were substantially higher in asthenospermia compared to oligoasthenoteratospermia (P ≤ 0.05). The mean percentages of histone transition abnormality and MEST methylation in oligoasthenoteratospermia were significantly higher than asthenospermia and normospermia (P ≤ 0.05). A negative correlation existed between the histone transition abnormality and MEST methylation with sperm parameters. In conclusion, chromatin integrity, sperm maturity, and MEST methylation may be considered important predictors for addressing male factor infertility. Therefore, we suggest that male infertility may be linked to methylation of the imprinted genes.
... In testicular sperm, methylation at the H19 DMR is especially susceptible to modification [51]. Interestingly, the genotype of an SNP in the H19 DMR affected nearby DNA methylation levels [52]. Epimutations in the H19 DMR have been detected in 20% of men with oligozoospermia, and paternally expressed gene 1/mesoderm-specific transcript DMR epimutations have been found in 3% of men with the condition [53]. ...
Infertility is defined as the failure to conceive after at least one year of unprotected intercourse. Long non-coding RNAs (lncRNAs) are transcripts that contain more than 200 nucleotides but do not convert into proteins. LncRNAs, particularly lncRNA H19, have been linked to the emergence and progression of various diseases. This review focuses on the role of H19 in infertility caused by polycystic ovary syndrome, endometriosis, uterine fibroids, diminished ovarian reserve, male factor, and assisted reproductive technology-related pathology, highlighting the potential of H19 as a molecular target for the future treatment of infertility.
... Furthermore, there was a significant reduction in H3K9 acetylation and H3K4 methylation compared to the fresh samples [165]. Another study showed that cryoprotectant and freezing-thawing protocols significantly increased global DNA methylation levels in ram spermatozoa [166]. Additionally, a study on humans by Khosravizadeh and co-authors investigated the effects of cryopreservation on DNA methylation in promoter regions of the SNURF-SNRPN and UBE3A imprinted genes, PWS-ICR, and AS-ICR in the chromosome 15q11-q13 region [167]. ...
... For example, CPA, a widely used agent for sperm cryopreservation, is cytotoxic and can harm sperm cells, causing osmotic injury and physiological alterations and potentially influencing the epigenetic state of sperm cells indirectly [149,[154][155][156][157]. Second, raising the level of ROS during the freezing-thawing process [156,[159][160][161] might induce site-specific hypermethylation through either the upregulation of DNA methyltransferases (DNMTs) or the formation of new DNMT-including complexes [158,159]. It is important to emphasize that sperm epigenetic changes could be secondary to additional factors, including sperm manipulation alone or patient characteristics [166][167][168][169][170]. For example, it is well known that oligospermic men have more epigenetic changes than normospermic men [160][161][162]. ...
Since the birth of the first baby by in vitro fertilization in 1978, more than 9 million children have been born worldwide using medically assisted reproductive treatments. Fertilization naturally takes place in the maternal oviduct where unique physiological conditions enable the early healthy development of the embryo. During this dynamic period of early development major waves of epigenetic reprogramming, crucial for the normal fate of the embryo, take place. Increasingly, over the past 20 years concerns relating to the increased incidence of epigenetic anomalies in general, and genomic-imprinting disorders in particular, have been raised following assisted reproduction technology (ART) treatments. Epigenetic reprogramming is particularly susceptible to environmental conditions during the periconceptional period and non-physiological conditions such as ovarian stimulation, in vitro fertilization and embryo culture, as well as cryopreservation procedure, might have the potential to independently or collectively contribute to epigenetic dysregulation. Therefore, this narrative review offers a critical reappraisal of the evidence relating to the association between embryo cryopreservation and potential epigenetic regulation and the consequences on gene expression together with long-term consequences for offspring health and wellbeing. Current literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, in terms of osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, it is therefore, critical to have a more comprehensive understanding and recognition of potential unanticipated iatrogenic-induced perturbations of epigenetic modifications that may or may not be a consequence of vitrification.
... Furthermore, there was a significant reduction in H3K9 acetylation and H3K4 methylation compared to the fresh samples [165]. Another study showed that cryoprotectant and freezing-thawing protocols significantly increased global DNA methylation levels in ram spermatozoa [166]. Additionally, a study on humans by Khosravizadeh and co-authors investigated the effects of cryopreservation on DNA methylation in promoter regions of the SNURF-SNRPN and UBE3A imprinted genes, PWS-ICR, and AS-ICR in the chromosome 15q11-q13 region [167]. ...
... For example, CPA, a widely used agent for sperm cryopreservation, is cytotoxic and can harm sperm cells, causing osmotic injury and physiological alterations and potentially influencing the epigenetic state of sperm cells indirectly [149,[154][155][156][157]. Second, raising the level of ROS during the freezing-thawing process [156,[159][160][161] might induce site-specific hypermethylation through either the upregulation of DNA methyltransferases (DNMTs) or the formation of new DNMT-including complexes [158,159]. It is important to emphasize that sperm epigenetic changes could be secondary to additional factors, including sperm manipulation alone or patient characteristics [166][167][168][169][170]. For example, it is well known that oligospermic men have more epigenetic changes than normospermic men [160][161][162]. ...
Since the birth of Louise Brown in 1978, more than nine million children have been conceived using assisted reproductive technologies (ARTs). While the great majority of children are healthy, there are concerns about the potential epigenetic consequences of gametes and embryo manipulation. In fact, during the preimplantation period, major waves of epigenetic reprogramming occur. Epigenetic reprogramming is susceptible to environmental changes induced by ovarian stimulation, in-vitro fertilization, and embryo culture, as well as cryopreservation procedures. This review summarizes the evidence relating to oocytes and embryo cryopreservation and potential epigenetic regulation. Overall, it appears that the stress induced by vitrification, including osmotic shock, temperature and pH changes, and toxicity of cryoprotectants, might induce epigenetic and transcriptomic changes in oocytes and embryos. It is currently unclear if these changes will have potential consequences for the health of future offspring.
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Maternally expressed 8 (MEG8) is a long non-coding RNA which is expressed in the nucleus. It is highly expressed in adrenal, placenta and brain. Recent studies have shown contribution of MEG8 in different disorders ranging from neoplastic ones to diabetic nephropathy, atherosclerosis, ischemic stroke, trophoblast dysfunction and abortion, Henoch-Schonlein purpura and osteoarthritis. It has an oncogenic role in the development of lung, pancreatic and liver cancer. In the current review, we summarize the role of this lncRNA in mentioned disorders, based on the evidence obtained from in vitro, in vivo and human studies.
... For instance, 3D morphology measurement instead of traditional fixation and staining can capture the changes of sperm structure more precisely [143]. To explore the impact on genetics, compared with DNA fragmentation measurement, telomere, DNA and RNA methylation, and histone modification analysis techniques are able to detect more specific and imperceptible genetic alterations [144][145][146]. The 24 h incubation post-ejaculation affected both sperm DNA methylation and integrity of mouse spermatozoa [146]. ...
Preservation of human spermatozoa in vitro at normothermia or hypothermia maintaining their functions and fertility for several days plays a significant role in reproductive biology and medicine. However, it is well known that human spermatozoa left in vitro deteriorate over time irreversibly as the consequence of various stresses such as the change of osmolarity, energy deficiency, and oxidative damage, leading to substantial limitations including the need for semen examinations, fertility preservation, and assisted reproductive technology. These problems may be addressed with the aid of non-freezing storage techniques. The main and most effective preservation strategies are the partial or total replacement of seminal plasma with culture medium, named as extenders, and temperature-induced metabolic restriction. Semen extenders consist of buffers, osmolytes, and antioxidants, etc. to protect spermatozoa against the above-mentioned adverse factors. Extended preservation of human spermatozoa in vitro has a negative effect on sperm parameters, whereas its effect on ART outcomes remains inconsistent. The storage duration, temperature, and pre-treatment of semen should be determined according to the aims of preservation. Advanced techniques such as nanotechnology and omics have been introduced and show great potential in the lifespan extension of human sperm. It is certain that more patients will benefit from it in the near future. This review provided an overview of the current knowledge and prospects of prolonged non-freezing storage of human sperm in vitro.
... Aberrant DNA methylation has been reported to be associated with male subfertility and abnormal semen parameters, and plenty of differential methylation sites or regions have been identified in several disease states, including teratozoospermia and oligozoospermia [35][36][37][38][39][40][41][42][43][44]. For instance, several candidate gene studies of DNA methylation have found an association of aberrant methylation of H19 with male infertility, including teratozoospermia and oligozoospermia [45,46]. A methylation study on the spermatogenesisassociated genes (SPATA4, SPATA5 and SPATA6) showed that hypermethylation of these genes correlated with oligozoospermic infertility [47]. ...
Aims & objectives: To investigate DNA methylation patterns in asthenozoospermic and normozoospermic sperm and to explore the potential roles of differential methylations in the etiology of the disease. Materials & methods: The authors performed whole-genome bisulfite sequencing analysis between normozoospermic controls and asthenozoospermic individuals. Results: The authors identified 238 significant differentially methylated regions. These differentially methylated regions were annotated to 114 protein-coding genes, with many genes showing associations with spermatogenesis, sperm motility etc. Conclusion: There are plenty of genomic regions exhibiting altered DNA methylation in asthenozoospermia, a number of which are located within or adjacent to sperm-related genes, suggesting novel methylation markers of asthenozoospermia and potential epigenetic regulation mechanisms through DNA methylation in the disease.
... In fact, several studies of differential DNA methylation have shown that the absolute difference in beta values of differential methylation sites is very small. For example, minor differences in beta values have been observed during fetal development (27), during aging of CD8 + T cells (28), and in race-associated methylation sites (29), along with many more examples from either array (30)(31)(32)(33)(34) or high-throughput sequencing (HTS) (35,36). Moreover, studies have revealed that DNA methylation states in different tissues are highly positively correlated (37)(38)(39). ...
Differential DNA methylation is a feature of numerous physiological and pathological processes. However, the extent to which single-base cytosine methylation modifies cellular responses to various stimuli has not been well characterized. In this study, we carried out a systematic analysis of methylome data derived from human blood and immune cells and constructed the ImmuMethy database. ImmuMethy allows interrogation of DNA methylation plasticity (MPL) at the single cytosine level. MPL, which refers to the variability of DNA methylation, is quantitatively measured in multiple ways, such as quartiles and standard deviations. ImmuMethy comprises over 36 000 samples from the Human Methylation450 and MethylationEPIC BeadChips platforms and provides multiple applications, such as an overview of methylation status and plasticity, differential methylation analysis, identification of methylation markers and sample stratification. An analysis of all datasets revealed that DNA methylation is generally stable, with minimal changes in beta values. This further supports the characteristics of DNA methylation homeostasis. Based on the beta value distribution, we identified three types of methylation sites: methylation tendency sites, unmethylation tendency sites and dual tendency or nonbiased methylation sites. These sites represent different methylation tendentiousness of DNA methylation across samples. The occurrence of multiple methylation tendencies in a site means split methylation, which generally corresponds to high MPL. Inverted methylation tendencies from methylation tendency sites to unmethylation tendency sites, or vice versa, represent strong differential methylation in response to conditions. All these sites can be identified in ImmuMethy, making it a useful tool for omics-based data-driven knowledge discovery.
Database URL: http://immudb.bjmu.edu.cn/immumethy/
... Так, анализ метилирования генов H19, GNAS, SNRPN и MEG8 в образцах спермы бесплодных мужчин с помощью метода таргетного бисульфитного высокопроизводительного секвенирования (RRBS, платформа Illumina) показал наличие 1,7 % метилированных по SNRPN образцов в группе мужчин с олигозооспермией против 0,42 % в группе контроля с нормозооспермией, хотя указанные различия в дифференциально метилированном районе SNRPN в цитируемой работе были недостоверны [26]. Можно предположить, что примесь геномной ДНК лейкоцитов могла способствовать появлению ложноположительных результатов ПЦР вследствие амплификации метилированных аллелей SNRPN материнского происхождения в настоящей работе. ...
Introduction. Male infertility is a common problem in andrology and occurs in 45 % of infertile couples. Some cases of male infertility caused by genetic reasons: point mutations at some monogenic diseases, AZF deletions or a CFTR mutation compounds; reduced fertility is also associated with polymorphic variants of the genes AR and GSTT1/GSTM1. At the same time, increasing amount of data are being published about the role of epigenetic mechanisms (aberrant methylation and imprinting alterations) in defective spermatogenesis.
Materials and methods. We have studied 49 sperm samples obtained from unrelated infertile men using polymerase chain reaction, fragment analysis, and sequencing.
Results. Five patients were excluded from the initial cohort: one with a repeat length of 29 (CAG) in the first exon of the AR, three with null genotypes in the GSTT1 and GSTM1, and one with the delF508/5T heterozygous compound in the CFTR. Thereafter, methylation of the imprinted gene SNRPN was determined using methyl-specific polymerase chain reaction and bisulfite sequencing. Aberrant SNRPN methylation was detected in 11.4 % of male infertility samples. In total, molecular genetic and epigenetic alterations were determined in 20 % of patients.
Conclusions. Obtained data demonstrate a significant proportion of (epi)genetic disorders in a heterogeneous cohort of men with reduced fertility.
