Evaluation of the expression levels of autophagy-related genes in patients with recurrent pregnancy loss compared to normal individuals

Abstract

Recurrent Pregnancy Loss (RPL) is one of the types of infertility disorders that occurs in 2-5% of women of childbearing age and various reasons have been mentioned for RPL, such as genetic, hormonal, anatomical, microbial and immunological disorders. The aim of this study is the evaluation of the expression levels of autophagy-related genes in patients with recurrent pregnancy loss compared to normal individuals. Materials and Methods: This research was a case-control study and the target population was women suffering from recurrent pregnancy loss who referred to Waliasr International Hospital in Tabriz in 2022. The final sample size in this study was 60 people, who were divided into two groups of 30 people: control (healthy women with a history of successful pregnancy) and case (women with recurrent pregnancy loss). The sampling method in this study was simple random. First, a written and informed consent was obtained from the target population. To prepare peripheral blood mononuclear cells (PBMCs) from peripheral blood, first 10 cc of heparinized blood were taken from patients and peripheral blood mononuclear cells were isolated using Ficol 1.077. The expression level of the studied genes was evaluated using Real Time-PCR technique. First, by using Trizol, all the RNAs in the cells were extracted, and then by using specific primers, cDNA was produced from the desired gene. Next, by using gene-specific primer and SYBR Green dye, which is an intercalating dye with double-stranded DNA, the cDNA synthesized from the said gene was amplified in a thermocycler and compared with the control group. In order to normalize the measurement of the mentioned genes, β-actin was used as a house keeping gene. Then the proteins were run on a 4-12% SDS-PAGE gel. The results of western blot were presented semi-quantitatively by densitometry method. The primers needed to check the expression of the desired genes were extracted from the reliable articles and checked with the help of available software, and then they were ordered to the reliable companies for manufacturing. Results: In this study, the expression levels of FOXO3a (P=0.003), LC3 (P=0.009), Beclin-1 (P=0.013) and Atg5 (P=0.039) genes in the group of women with recurrent pregnancy loss was significantly lower than in women with a history of successful pregnancy. Also, with the help of SDS-PAGE method, it was determined that the peptide expression level of FOXO3a, LC3, Beclin-1, Atg5 and β-Actin genes was clearly lower in the group of women with recurrent pregnancy loss than in women with a history of successful pregnancy. The peptide expression level of FOXO3a (P=0.017), LC3 (P=0.01), Beclin-1 (P=0.034) and Atg5 (P=0.040) genes in the group of women with recurrent pregnancy loss was significantly lower than in women with a history of successful pregnancy.