This study aimed to validate two DNA extraction methods collected from buccal cells to gain a genomic DNA and to determine the effect of process time (from collection time to amplification). Total number of 116 buccal swab samples from unrelated healthy volunteers were subjected to DNA extraction by using two methods: modified (phenol chloroform) (Organic) and Prep Filer Forensic DNA Extraction kit. DNA was evaluated according to the: quantity, integrity and suitability for genetic analysis. The DNA yield and purity were measured by Nano drop Spectrophotometer. Organic method was modified from the classical method (addition Dithiothreitol to lysis buffer), The purity average values increased from 1.4 to 1.6 and it had the highest average concentration (73.3ng/µl) while the highest purity was observed in Prep Filer Forensic DNA Extraction kit with average (1.82). Delayed in sample processing effect both quantity and integrity of the extracted DNA. All samples were genetically analyzed at 16 STR with amelogenin (DNA markers) included in the Amp FlSTR® Identifiler™ PCR Amplification Kit panel from Applied Biosystems. Buccal cell swabs were very good source of genomic DNA especially for large population studies.
In the vast majority of instances, the causes of male infertility are unknown, although scientists have lately attempted to correlate epigenetics to infertility mechanisms. DNA methylation is an important biological modification that gives phenotypic diversity and adaptability. The aim of this study was whether the methylation status of the methylenetetrahydrofolate reductase (MTHFR) gene promoter in Iraqi men’s spermatozoa is correlated with male infertility. Semen samples were collected from 100 infertile and healthy fertile men. Divided into 4 groups included 25 subjects of healthy fertile men as a control group(C) and infertile groups, which divided into (3) subgroups: Asthenozoospermia (A) (n=25), Oligoasthenozoospermia (OA) (n = 25) and severe Oligoasthenoteratozoospermia (SOA) (n =25). Sperm DNAs and RNAs from all samples were isolated; followed by sperm-specific methylation detection using the EpiJET DNA Methylation Analysis Kit, which is simple digestion was performed with two restriction enzymes (MspI/HpaII). The methylation percentage (mean ± SD) of the MTHFR gene promoter was quantified in each studied group as follow:[ (C) ( 1.76±0.46) (A) (3.59±0.89), (OA) (26.10±4.14) and (SOA)( 29.13±4.88)]. The percentage of MTHFR promoter methylation infertile men was significantly higher than that in the healthy control group (p = 0.00). Methylation level was determined via receiver operator characteristic (ROC) analysis as the cut-off values of each infertile subgroup were as follows: [(A) (2.43%), (OA) (7.59%) and (SOA) (10.96%)]. According to WHO guidelines, sperm concentration, progressive motility and morphologically normal sperm were determining (WHO, 2010). The qRT-PCR was used to evaluate MTHFR gene expression in all sperm samples. The results revealed a substantial reduction in fold expression (2–∆∆Ct) in all infertile groups when compared to the control g roup, with a P-value (0.002). In this research findings suggest that epigenetic impairment (hypermethylation) of the MTHFR promoter region in sperm DNA from OA and SOA patients may increase male infertility risk.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.