Peripheral blood samples (2 mL) were obtained from varicocele patients and the DNA was extracted using a standard salting-out procedure ( 19). The examination of blood samples was approved by the local ethics review. All participants were fully informed of the objectives of the study and those that signed the consent form were assigned to the study. The control group (healthy volunteers) consisted of 80 fertile and normospermic men from Yazd Infertility Center who fathered at least one child. Patients with varicocele were divided into three grades: Grade I (n = 13), Grade II (n = 31) and Grade III (n = 38). Semen analysis was performed according to the WHO laboratory manual ( 18). Non-obstructive azoospermic individuals were not included in this study. Physical examination in standing position and via scrotal palpation in a temperature controlled room (23 ☌) was carried out to confirm varicocele. A deliberate mismatch at position -2 or -3 from the 3' terminal end of the inner primers can improve allele specificity ( 16).Ī case-control study of 82 male patients with clinical varicocele and 80 male controls (healthy volunteers) were recruited from the Yazd Infertility Center ( 17). The product can easily be discriminated on gel electrophoresis either as homozygous or heterozygous. The outer primers amplify a large fragment of the target gene contains variant nucleotide as a control fragment and smaller allele-specific amplicons with different sizes. In T-ARMS-PCR, a pair of common (outer) primers produces a non–allele-specific PCR product and in combination with two inner, allele-specific primers (in opposite orientation to each other) produces 2 PCR products. Here in T-ARMS-PCR, both normal and mutant alleles can be run altogether with a control fragment in a single reaction. In conventional ARMS-PCR, the amplification of normal and mutant allele is carried out in two separate reactions. These techniques are relatively slow and very expensive in comparison to Tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR) ( 12- 15). So far genotyping of G894T has been performed via polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), real time-PCR and direct sequencing. Several reports are indicative of reduced eNOS activity in substituted individuals ( 11). The common eNOS source of polymorphism observed in various populations is G894T in exon seven that leads to Glu298Asp, ( 9). NO is being produced by endothelial nitric oxide synthase (eNOS) ( 6- 8). Nitric oxide (NO) is an important antioxidant found in seminal plasma ( 5). ROS may damage morphology, motility and sperm concentrations, leading to loss of fertility. Although the pathogenic mechanisms by which varicocele leads to changes in spermatogenesis are not clear, some of these mechanisms may possess a known cause, such as generation of reactive oxygen species (ROS) ( 3, 4). Varicocele occurs in approximately 30-40% of infertile males ( 2). A majority of idiopathic varicocele generally occurs on the left side ( 1). Inflammation of the pampiniform venous plexus in scrotum is the main identifiable causes of varicocele. Full accordance between PCR-RFLP and T-ARMS-PCR methods for genotyping of rs1799983 polymorphism was found.Ĭonclusions: This is the first work that describes a rapid, relatively cheap, high throughput detection of G894T polymorphism in eNOS that can be used in large scale clinical studies. Results: The results showed that GG (varicocele infertile men), GT and TT genotypes appear to be 53.65%, 34.14%, and 12.19%, respectively. Materials and Methods: A T-ARMS-PCR for rs1799983 polymorphism in a single-step PCR was carried out, and the results were confirmed by PCR-RFLP technique in 82 infertile men with varicocele. Objectives: Development of a new Multiplex Tetra-Primer Amplification Refractory Mutation System - Polymerase Chain Reaction (T-ARMS-PCR) for detection of rs1799983 (G894T) in the human eNOS was sought. Background: The transversion of G to T (G894T) in human endothelial nitric oxide synthase ( eNOS) gene has profound effects such as male infertility, recurrent miscarriage, multiple sclerosis and cardiovascular diseases.
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