Sperm chromatin and DNA integrity, methyltransferase mRNA levels, and global DNA methylation in oligoasthenoteratozoospermia / 대한생식의학회지

OBJECTIVE: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. METHODS: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. RESULTS: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. CONCLUSION: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

SULF 1 gene polymorphism, rs6990375 is in significant association with fetus failure in IVF technique

Sulfatase 1 [SULF1] function is to remove the 6-O-sulphate group from heparan sulfate. This action changes the binding sites of extracellular growth factors. SULF1 expression has been reported to be changed in angiogenesis. We hypothesized that single nucleotide polymorphisms [SNPs] of SULF1 would impact clinicopathologic characteristics. Study of SULF1 gene polymorphism with fetus failure in in vitro fertilization [IVF] technique. We studied one common [minor allele frequency >0.05] regulatory SNP, rs6990375, with polymerase chain reaction and restriction fragment length polymorphism method, in 53 infertile women with fetus failure in IVF technique and 53 women with at least one healthy child as controls. We found that rs6990375 is significantly associated with an early failure in IVF and frequency of G allele is high in women with fetus failure in IVF technique [p<0.001]. These findings suggest that SULF1genetic variations may play a role in IVF technique fetus failure. Further studies with large sample sizes on SULF1 SNPs may be useful in support of this claim

cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles [NPs] and 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol [PEG], DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs [P<0.05]. Hydroxyapatite NPs conjugated with DOPE are a good choice for gene delivery and silencing of viral genes in cervical cancer cells, but their efficacy should be addressed more in future studies