The present study was approved by the Human Ethics Research Committee of the Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences.
All of the controls and subjects who agreed to participate in this study have signed informed consent before the collection of peripheral blood samples.
One hundred women with idiopathic spontaneous repeated pregnancy loss who were attending the recurrent miscarriage clinic and 100 women without any miscarriage who had at least one successful pregnancy were recruited in this study. Control blood samples were collected in routine checkup test.
Blood collection and DNA extractions
Five ml peripheral blood samples were collected in blood collection tubes containing EDTA for anticoagulation. They stored at -20°c until further used for DNA extraction. Genomic DNA extraction was carried out using a commercially available kit (Qiagen, Germany. cat.No.51104) according to the manufacturer’s protocol.
The quality of DNA was assessed using 1% agarose gel electrophoresis. Concentration and purity of DNA were estimated in absorbance at 260 nm by spectrophotometer (thermo scientific).
Polymerase chain reaction analyses (PCR)
The length of the target gene in our study was 180 bp and the genomic DNA was amplified by the following primer sequences. Besides, the Tm temperature was considered at 68°C.
Forward primer; 5'-TGGAGAGTGCTGGTGTACCCCA- 3'
Reverse primer; 5'-GCCTCCACCCCCACCCTGTC- 3'
Optimized PCR reaction was performed in a total reaction volume of 25 µl containing: 12/5 µl PCR master mix (2X), 1 µl forward primer (10 pmol), 1 µl reverse primer (10 pmol), 3 µl genomic DNA (50 ng), and 7/5 µl distilled water. Finally, the tubes were placed onto a thermocycler (Astec-Japan). Cycling conditions were as follows: initial denaturation at 95 °C for 10 min; followed by 35 cycles of amplification; 95 °C for 15 s; 68 °C for 15 s; 72 °C for 30 s. The final extension was done at 72 °C for 5 min. Amplification of PCR products were also confirmed by gel electrophoresis (1/5% agarose gel).
Final assessing was performed by PCR-RFLP method. In other words, digestion of the PCR product with the restriction enzyme NgoMIV allowed detection of the alleles of -786T > C. this enzyme cleavage site on the gene is shown in Fig. 1. The digestion reaction was carried out in a total volume of 25 µl, containing 10 µl PCR product, 0/25 µl R buffer (10X), 0/25 µl NgoMIV enzyme, and 14/5 µl distilled water. The tubes were incubated at 37°C for 3 hours. After digestion, the samples were separated by gel electrophoresis (2% agarose gel) so as to visualize the different products.
All data were analyzed using the SPSS standard software (Version 21.0, IBM, Armonk, NY, USA) and chi-square test. Additionally, the results were reported as a graphical representation by GraphPad Prism 6 software. P-values of < 0.05 were considered statistically significant for all data.