Comparative Epigenetic Analysis of Imprinting Genes Involved in Fertility, in Cryopreserved Human Sperms with Rapid Freezing versus Vitrification Methods.

OBJECTIVE: Choosing the optimal method for human sperm cryopreservation seems necessary to reduce cryoinjury. The aim of this study is to compare two cryopreservation methods including rapid-freezing and vitrification, in terms of cellular parameters, epigenetic patterns and expression of paternally imprinted genes (PAX8, PEG3 and RTL1) in human sperm which play a role in male fertility. MATERIALS AND METHODS: In this experimental study, semen samples were collected from 20 normozoospermic men. After washing the sperms, cellular parameters were investigated. DNA methylation and expression of genes were investigated using methylation-specific polymerase chain reaction (PCR) and real-time PCR methods, respectively. RESULTS: The results showed a significant decrease in sperm motility and viability, while a significant increase was observed in DNA fragmentation index of cryopreserved groups in comparison with the fresh group. Moreover, a significant reduction in sperm total motility (TM, P<0.01) and viability (P<0.01) was determined, whereas a significant increase was observed in DNA fragmentation index (P<0.05) of the vitrification group compared to the rapid-freezing group. Our results also showed a significant decrease in expression of PAX8, PEG3 and RTL1 genes in the cryopreserved groups compared to the fresh group. However, expression of PEG3 (P<0.01) and RTL1 (P<0.05) genes were reduced in the vitrification compared to the rapid-freezing group. Moreover, a significant increase in the percentage of PAX8, PEG3 and RTL1 methylation was detected in the rapid-freezing group (P<0.01, P<0.0001 and P<0.001, respectively) and vitrification group (P<0.01, P<0.0001 and P<0.0001, respectively) compared to the fresh group. Additionally, percentage of PEG3 and RTL1 methylation in the vitrification group was significantly increased (P<0.05 and P<0.05, respectively) compared to the rapid-freezing group. CONCLUSION: Our findings showed that rapid-freezing is more suitable method for maintaining sperm cell quality. In addition, due to the role of these genes in fertility, changes in their expression and epigenetic modification may affect fertility.

The Nsun7 (A11337)-deletion mutation, causes reduction of its protein rate and associated with sperm motility defect in infertile men.

PURPOSE: Recent studies have shown that genetic abnormalities may be responsible for most unknown cases of male infertility. Human Nsun7 gene, which is located on chromosome4, has a role in sperm motility by encoding the putative methyltransferase Nsun7 protein. The aim of the present study was to investigate the mutations of exon4 in the Nsun7 gene, which is associated with sperm motility defect. METHODS: Semen samples including those of fertile normospermic (normal), infertile oligospermic (with normal sperm motility), and infertile asthenospermic (with reduced sperm motility) men were collected from the Omid and Fatemezahra IVF centres (Babol, Iran). These samples were then analysed on the basis of World Health Organization guidelines using the general phenol-chloroform DNA extraction method. Exon4 was amplified using Sun-F/Sun-R primers. Samples from asthenospermic men, which showed different patterns of movement on single-strand conformation polymorphism compared with normal and oligospermic samples, were identified and subjected to sequencing for further identification of possible mutations. RESULTS: Analysis of extracted sperm proteins showed that the rate of Nsun7 decreased. Likewise, direct sequencing of PCR products, along with their analysis, confirmed the deletion mutation of adenine in location 11337 of the Nsun7 gene in asthenospermic men. Comparison of normal and mutant protein structures of Nsun7 indicated that the A11337-deletion of the exon4 resulted in the valine residues-157 with GTA-codon in normospermic replaced with TAG-early stop codon in asthenospermic samples, causing an abortive protein product with amino acid sequence shorter than normal. The secondary structure of the protein, the protein folding, and ligand binding sites were changed, indicating the impairment of the protein function. CONCLUSIONS: Because the Nsun7 gene products have a role in sperm motility, it will lead to impairment in the activity of the protein and motility of sperm flagella as well as male infertility if a mutation occurs in this gene.

T26248G-transversion mutation in exon7 of the putative methyltransferase Nsun7 gene causes a change in protein folding associated with reduced sperm motility in asthenospermic men.

The NOP2/Sun domain family, member 7 (Nsun7) gene, which encodes putative methyltransferase Nsun7, has a role in sperm motility in mice. In humans, this gene is located on chromosome 4 with 12 exons. The aim of the present study was to investigate mutations of exon 7 in the normospermic and asthenospermic men. Semen samples were collected from the Fatemezahra IVF centre (Babol, Iran) and analysed on the basis of World Health Organization (WHO) guidelines using general phenol-chloroform DNA extraction methods. Exon 7 was amplified using Sun7-F and Sun7-R primers. Bands on samples from asthenospermic men that exhibited different patterns of movement on single-strand conformation polymorphism gels compared with normal samples were identified and subjected to sequencing for further identification of possible mutations. Direct sequencing of polymerase chain reaction (PCR) products, along with their analysis, confirmed C26232T-transition and T26248G-transversion mutations in asthenospermic men. Comparison of normal and mutant protein structures of Nsun7 indicated that the amino acid serine was converted to alanine, the structure of the helix, coil and strand was changed, and the protein folding and ligand binding sites were changed in samples from asthenospermic men with a transversion mutation in exon 7, indicating impairment of protein function. Because Nsun7 gene products have a role in sperm motility, if an impairment occurs in exon 7 of this gene, it may lead to infertility. The transversion mutation in exon 7 of the Nsun7 gene can be used as an infertility marker in asthenospermic men.