Expression of genes involved in the inflammatory response in human granulosa cells in short-term in vitro culture

Abstract The essential function of granulosa cells is to maintain the proper course of oogenesis and folliculogenesis. The immune system is an additional local regulator of ovarian function, with cytokines necessary for the proper function of the ovaries, including the secretion of steroid hormones This study aimed to analyze the expression of genes in human GCs in short-term primary culture and define the difference in the expression of IL1β, IL6, and TNFα genes at 48h and 72h of culture compared to the 24h control. Total RNA was isolated using the Chomczyński and Sacchi protocol. RNA samples were treated with DNase I and reverse transcribed (RT) into cDNA. The determination of transcript levels of the mentioned genes was performed using the Light Cycler® 96 Real-Time PCR kit, Roche Diagnostics GmbH (Mannheim, Germany). The present study proved that granulosa cells in a short-term primary in vitro culture express IL-1β, IL-6, and TNFα. The tested genes show a decrease in expression at 24h of culture and a subsequent slight increase at 72h, not exceeding the initial levels. The expression changes the most for IL1β and the least for TNFα. The fluctuations in the amount of transcript may be influenced by factors stored in granulosa cells before the IVM procedure, the procedure of in vitro fertilization, as well as factors related to the process of primary culture. More research is needed to understand the details of these occurrences. Running title: The inflammatory response in human granulosa cells