P-661 Comparative and descriptive molecular analysis of cumulus and luteinized mural granulosa cells for steroidogenic function in IVF patients

Abstract

Abstract Study question Do cumulus granulosa cells (CCs) have steroidogenic function and gonadotropin response like luteinized mural granulosa cells (LMGCs)? Summary answer Yes. Both types of granulosa cells have similar expression levels for the steroidogenic enzymes and hCG/LH receptors. What is known already CCs and LMGCs have distinct roles in follicular development, oocyte growth and maturation, cumulus expansion, ovulation, and luteinization. CCs support oocytes, especially during meiotic resumption, ovulation, and fertilization. They exhibit specialized functions in response to hormones like LH and participate in bidirectional communication with the oocyte. Although LMGCs are primarily responsible for steroidogenic function and progesterone (P4) production, very limited data is available as to whether CC themselves express the steroidogenic enzymes and gonadotropin receptors and produce sex steroids at basal or after gonadotropin stimulation in humans. We aimed to address this issue in the current study. Study design, size, duration An in-vitro basic science study was designed to compare CCs and LGMCs in the context of vitality, steroidogenic capabilities (both in baseline and response to rec-hCG (10 IU/ml), and EGF pathway responses. The assessment was planned to compare hormonal assay in spent culture and transcriptional levels. The study’s duration encompassed the period of ovarian stimulation, oocyte retrieval, and subsequent cellular analysis. Participants/materials, setting, methods 24 IVF patients undergoing ovarian stimulation with progestin primed (PPOS) and GnRH antagonist protocols for different infertility etiologies were included. Ovarian stimulation and ovulation trigger were done using rec-FSH and rec-hCG, respectively. CCs were collected from the denudation plate. LMGCs were obtained from follicular aspirates during the OPU and cultured individually for each patient without pooling in culture plates. After overnight incubation, the cells were treated with rec-hCG overnight. RNA isolation was performed. Main results and the role of chance The expression of the steroidogenic enzymes (StAR, SCC, 17B-HSD, 3B-HSD, Aromatase), LH receptor and EGF-like amphiregulin (AREG) in CCs were comparable to LMGCs on quantitative RT-PCR analysis. In line with this finding, we found they produced similar amounts of estradiol (E2) and progesterone (P4) in culture. Furthermore, similar to LMGCs, hCG treatment of the CCs resulted in a significant increase in P4 output, indicative of the presence of functional LH receptors. When the culture period was extended to six days, CCs retained their vitality and steroidogenic function like LMGCs and produced detectable E2 and P4. Limitations, reasons for caution Caution should be exercised when interpreting our data as it is unclear if the parameters analyzed here vary according to the infertility etiology, stimulation protocol, mode of trigger and any underlying disease that concomitantly present. Wider implications of the findings Prominent steroidogenic activity of CCs might have other important implications for a successful pregnancy. Moreover, similar expression profiles of steroidogenic pathways and hCG/LH receptors between CCs and LMGCs indicate that CCs can also be used to understand better the luteal function and intricate crosstalk between in oocyte microenvironment. Trial registration number N/A