Adjuvant recombinant Lh(rLH) or growth hormone (GH) to the antagonist protocol in poor responders undergoing IVF

Abstract

in the human ovary remains unknown. The purpose of our study was to determine the role of hMSC secretome on immortalized unluteinized gonadotropin dependent human granulosa cell line (GC). DESIGN: Prospective in vitro studies using immortalized unluteinized gonadotropin dependent human granulosa cell line. MATERIALS AND METHODS: GCs were seeded on culture dishes precoated with extracellular matrix at a density of 6x104 cells/ml and cultured for 24 hours before treatment. Media was then collected, and either replaced by control media (C) or conditioned media (Cm). Cm was previously prepared by collecting the media of hMSC when those reached 90% confluence in their culture plate. A subset of those two groups was treated with recombinant follicle stimulating hormone (FSH) at a concentration of 50 ng/ml. Forty eight hours after media change and FSH treatment, cells and supernatant were collected for analysis. Proliferation assay based on Ki67 analysis with flow cytometry was performed. Genes mRNA and protein expression levels were quantified by real-time PCR and Western blot, respectively. Estrogen levels were measured by ELISA. RESULTS: Human GCs cultured in hMSC Cm showed significantly higher proliferation rates when treated with Cm compared to the control group based on the percentage of gated Ki67 + cells (4.02 % AE0.78 vs 30.9 AE 4.62, P<0.001). The addition of FSH to both groups showed a trend of increased proliferation in the Cm group, however this did not reach statistical significance (52.7 AE 2.45 vs 53.1 AE 2.6, P>0.05). Human aromatase mRNA expression was 20 fold increased with Cm compared to the C group (P < 0.05), and 30 fold increase with CmF compared to the CF group (P < 0.05). This was also shown at the level of human StAR mRNA without FSH (14 fold increase, P<0.05), and with FSH (30 fold increase, P < 0.05). Protein studies and other gene expression profiles, in addition to results with molecular pathway inhibitors are being analyzed and will be presented during the meeting in October 2018. CONCLUSIONS: These findings show, for the first time, that hMSC secretome promotes human granulosa cell proliferation and regulates gene expression involved in folliculogenesis, such as aromatase. Further investigation, including co-culture and analysis of Cm is warranted to fully understand the effect of hMSC on human granulosa cells.