مقالات پذیرفته شده در هفتمین کنگره بین المللی زیست پزشکی
The Application of Gene Expression Analysis by ART in the Selection of Competent Genes for Pregnancy Prediction
The Application of Gene Expression Analysis by ART in the Selection of Competent Genes for Pregnancy Prediction
Ali Akbari,1,*Sayed Mehrdad Azimi,2
1. Department of Anatomical and Molecular Biology Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 2. Department of Anatomical and Molecular Biology Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Introduction: One of the most important steps in human assisted reproductive technology (ART) is selecting high-quality embryos for transfer to ensure successful outcomes. Traditionally, assessment of physical characteristics of the oocyte and embryo famed as morphological measures were reliable but it cannot fully determine the developmental potential of an embryo. To bypass limitations, biological measures such as gene expression profiles of cumulus cells (CCs) to enhance the accuracy of embryo selection were developed. It is possible to gain insights into the embryo's pregnancy potential by analyzing the gene expression patterns of these cells which surround the oocyte. Among of diseases that can deteriorate fertility, polycystic ovary syndrome (PCOS) affects the ovary's function, hormonal balance, and can alter the gene expression profile of CCs leading to irregular ovulation and potential difficulties in conceiving. Studies have shown some specific genes (e.g., CALM, PSMD6, and AK124742) are suggested for predicting pregnancy and were differentially expressed in the CCs of pregnant patients compared to the non-pregnant group and in PCOS and non-PCOS ovaries. This study was designed to explore the candidate genes for the selection of the most viable embryo.
Methods: Cross-sectional and 3-week prospective study was conducted between April 2020 and January 2021 at Hazrat Maryam Fertility Center of Shahid Beheshti hospital. This study investigated associations of infertility with gene expression among middle-aged Iranian adults with an average age of 32.48 years. Over all 66 patients (33 in the control group and 33 in the PCOS Group) participated who underwent an In vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatment participated. In normal ICSI or IVF cycles, a part of the CCs separated from the cumulus-oocyte using two sharp needles and were rapidly transferred into a tube containing a free enzyme medium. All of the collected CCs were washed with PBS and after centrifugation at 12,000 g for 2 minutes, The CCs mass of each patient was quickly stored in a -80 ° C freezer for Real-time PCR assessment of CALM, PSMD6, and AK124742. Glyceraldehydes-3- phosphate dehydrogenase (GAPDH) was used as an endogenous control. The expression level of each target gene was calculated as 2−ΔΔCt. Statistical analysis was performed using SPSS 20.00 Quantitative variables using mean and standard deviation (SD). The results were analyzed by the independent sample t-test and One-way ANOVA. The nominal and frequency data were analyzed using Chi-square test or Fisher’s exact test as appropriate. The Pearson correlation coefficient was used to examine the parametric variables (for nonparametric variables, Spearman's correlation coefficient was used). P values less than 0.05 were considered statistically significant for all the statistical tests.
Results: Expression of CALM1, PSMD6, and AK124742 revealed differential regulation in the pregnant group and the non-pregnant group. CALM1 showed a modest upregulation in the pregnant group, this increase did not reach statistical significance. In contrast, PSMD6 (p <0.001) and AK124742 (p <0.05) demonstrated significantly higher expression levels in pregnant compared to non-pregnant. The expression of CALM1 and AK124742 genes increased significantly and the expression of PSMD6 significantly downregulated in PCOS group compared to the control group (p <0.05).
Conclusion: These three specific genes in CCs have crucial role in pregnancy. PSMD6 can encode a regulatory subunit of the proteasome which degrades intracellular proteins and its activity may be increased to handle changing demands on protein metabolism in pregnant women. Though the function of AK124742 is unknown, its significant upregulation points to a potential role during pregnancy which needs further investigation. CALM1 encodes calmodulin for essential calcium signaling in ovulation and uterine contraction. Its upregulation could reflect perturbations in calcium signaling contributing to the infertility associated with PCOS. The different regulation of AK124742 and PSMD6 in pregnancy and PCOS provides evidence that PCOS specifically disrupts the gene regulatory networks induced during normal pregnancy. It is suggested that selecting genes that affect the reproductive process without careful examination of the path of the effect of these genes and only relying on comparing the expression of these genes in fertile and infertile groups cannot be an accurate method for selecting these genes.