FRI410 ESR1 Variants Reduce Estrogen Receptor Alpha Expression In Women With Unexplained Infertility

Disclosure: R.A. Roman: None. H. Liu: None. L.P. Chorich: None. Y. Li: None. J.E. Hall: None. M.P. Diamond: None. K.S. Korach: None. L.C. Layman: None. Objective: Estrogen receptor alpha (ERα), encoded by ESR1, is vital to human reproduction. DNA sequencing previously identified four ESR1 missense variants of uncertain significance in 197 cisgender women with well-characterized unexplained infertility, including a p.Thr313Met variant with impaired estrogen signaling in vitro. The p.His6Tyr, p.Ser118Pro, and p.Arg269Cys ESR1 variants did not affect estrogen signaling. We hypothesize that milder ESR1 variants could decrease ERα expression in women with unexplained infertility. Methods: Clinically identified ESR1 variant plasmids were constructed using site-directed mutagenesis and confirmed by Sanger sequencing. HepG2 cells were transiently transfected with either empty vector, wild type (WT) ESR1, p.His6Tyr, p.Ser118Pro, p.Arg269Cys, p.Thr313Met, or p.Gln375His (ESR1 missense variant control) estrogen receptor variant expression plasmids using Lipofectamine 3000 transfection reagent. Cell lysis was isolated from HepG2 cells 24 hours after transfection using RIPA lysis buffer. ERα expression was assessed by western blot fluorescence imaging and normalized to β-actin on LI-COR Odyssey DLx. Quantitative analysis was performed using Empiria Studio Software v.2. Results: Western blot analysis showed that the ESR1 p.His6Tyr variant had 87% decreased and the p.Ser118Pro variant had 75.6% decreased expression compared to WT ERα in vitro. The p.Arg269Cys, p.Thr313Met, and p.Gln375His variants had comparable expression to WT. Conclusion: Although the p.His6Tyr and p.Ser118Pro variant receptors have conserved estrogen signaling, our preliminary data show that they decrease ERα expression in vitro. This could be an underlying pathophysiologic mechanism affecting some women with unexplained infertility. Additional in vitro and in vivo analyses can further characterize their pathogenicity. Presentation: Friday, June 16, 2023