Methylation-specific polymerase chain reaction (MSP)

Genomic DNA was extracted from COCs by the QIAamp DNA Micro Kit (Cat NO: 56304, QIAGEN, Netherlands) according to the manufacturer’s instructions. DNA quantity and quality were measured using a Nanodrop 2000 Spectrophotometer (Thermo, USA). Genomic DNA was treated by bisulfite using the EZ DNA Methylation-Gold (Zymo Research, Germany) according to the manufacturer’s instructions. After conversion, DNA was eluted in buffer (Qiagen, Germany) to a final concentration of 30 ng/μl.

The DNA methylation status of ICAM-1 and HLA-G genes in COCs samples was evaluated by the methylation-specific PCR (MSP). For MSP, we had to use specific primer pairs for both methylated and unmethylated promoter sequences. The primers were designed using the Meth primer and GeneRunner software. Each MSP reaction was performed in a total volume of 25 μL. One microliter of sodium bisulfite converted DNA was added into a 24 μL reaction mixture containing 0.5 U of hot start Gold Taq Polymerase (Cat NO: M5001, Promega, USA), 5 μL of the 10× PCR buffer, 2.0 μL of MgCl₂ (50 mmol/L), 0.5 μL of dNTP (10 mmol/L; Fermentas, USA) and 1 μL of the corresponding forward and reverse primers (10 μmol/L dH2O up to final volume of 25 μL). Sodium bisulfite treated DNA was amplified in two separate MSP reactions, one with a set of primers specific for methylated, and the one for unmethylated ICAM-1 and HLA-G promoters sequences (Table ​(Table1).1). For MSP positive control, we used fully methylated DNA (100%) and unmethylated DNA (0%), which the fully methylated DNA was made of MsssI methylase (NEB, Ipswich, MA, USA) using human placental genomic DNA (gDNA; Sigma Aldrich, USA), and the unmethylated DNA was made of human placental genomic DNA (gDNA; Sigma Aldrich, USA). For MSP method sensitivity, we used the fully methylated and unmethylated DNA (EpiTect Control DNA and Control DNA Set, Qiagen, Germany) with different serial dilutions: the ratio 0% to 100%, 5% to 95%, 10% to 90%, 20% to 80%, 35% to 65%, 50% to 50%, 75% to 25% and 100% to 0% unmethylated DNA was combined with methylated DNA, respectively. Thermo cycling conditions were as follows: 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, 63 °C for 60 s and 72 °C for 60 s, with a final extension of 72 °C for 4 min. MSP products for methylated and unmethylated ICAM-1 and HLA-G promoters were run on 2% agarose gels containing 40 mMTris-acetate/1.0 mM EDTA (pH = 8) and were visualized by ethidium bromide staining (Fig. 2). Methylation genes pattern visualization was performed using the enhanced chemiluminescence detection system (UVITEC, UK). The intensity of each MS-PCR bands on the agarose gel was quantified using the ImageJ software version 1.50i (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).

Methylation profiling of HLA-G and ICAM-1 by methylation-specific PCR (MSP). Results of MSP on HLA-G and ICAM-1 promoters from gDNA isolated from with healthy lifestyle habit and without healthy lifestyle habit of POR patients. (M = methylated primer, UN = unmethylated primer). The 1 and 2 bands are for MSP positive control of UN methylated gDNA (10%). The 7 and 8 bands are for MSP positive control of UN methylated gDNA (20%). The 13 and 14 bands are for MSP positive control of fully methylated placental gDNA (100%). The 3, 4, 9 and10 bands are for HLA-G and the 5, 6, 11 and12 bands are for ICAM-1. The (3, 4) and (5, 6) bands are for POR patients with healthy lifestyle habits and (9, 10) and (11, 12) bands are for POR patients without healthy lifestyle habit. Columns 1, 3, 5, 7, 9, 11 and 13 belong to the patient sample with unmethylated primer and columns 2, 4, 6, 8, 10, 12 and 14 belong to the patient sample with methylated primer. Sample No. 1, 2, 7, 8, 13, and 14 bands are positive controls (DNA samples of fertile women). Sample No. 3, 4, 5, 6, 9, 10, 11, and 12 belong to different patient samples.