Effect of bisphenol A on alterations of ICAM-1 and HLA-G genes expression and DNA methylation profiles in cumulus cells of infertile women with poor response to ovarian stimulation

Abstract

This study aimed to investigate the relationship between follicular fluid Bisphenol A (BPA) concentrations with alterations of ICAM-1 and HLA-G genes and proteins expression as well as methylation profiles in the cumulus cells of poor ovarian response (POR) women based on their healthy lifestyle habit. Eighty women under the age of 35 were divided into two groups: 1-POR without using plastic containers (n = 40) and 2-POR with using plastic containers (n = 40). The ICAM-1 and HLA-G genes and protein expressions were examined by the quantitative PCR and western blotting technique. The methylation pattern was investigated by the methylation-specific PCR. Total BPA in follicular fluid was measured with high-performance liquid chromatography technique and the detection limit was 1.14 ng/ml. ICAM-1 and HLA-G genes were differentially expressed between the two groups studied. ICAM-1, HLA-G genes, and protein expressions in group 1 were up-regulated compared to the second group (P < 0.05). While DNA methylation status in group 1 were decreased compared to the other group (P < 0.05). The concentration of BPA in the follicular fluid of group 1 was lower compared to the second group (P < 0.05). The oocyte quality and clinical pregnancy ratio showed significantly higher in group 1 than in the other ones (P < 0.05). The alteration of ICAM-1 and HLA-G gene expressions in POR women is probably related to BPA concentration. As a result Lifestyle habits may also affect the methylation pattern and protein levels in the cumulus cells of POR women. Additionally, lifestyle habits may be considered as a marker for ovulation, oocyte maturation, preimplantation, and clinical pregnancy process.

Figure 1

Comparison of (a) HLA-G (43 kDa), (b) ICAM-1 (100 kDa), and (c) β actin protein as control. Protein levels in western blot analysis were examined between POR patients without healthy lifestyle habit and with healthy lifestyle habit. In the (a) and (b) pictures, WH lanes belong to the POR patients with healthy lifestyle habit and WOH lanes belong to the POR patients without healthy lifestyle habit (Supplementary Information).

Figure 2

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.

Figure 3

(A) Comparison of ICAM-1 and HLA-G genes expression of q-PCR analysis. (B) ICAM-1 and HLA-G proteins expression and, (C) ICAM-1 and HLA-G DNA methylation status between without healthy lifestyle habit (WOH) and with healthy lifestyle habit (WH) of POR patients. *P < 0.05, **P < 0.01, ***P < 0.001 was considered significant. N = 40, (40 patient in each group was assessed).