We appreciate your review of our revised manuscript, constructive comments, and suggestions for improving the manuscript. We deeply understand the significance of the experiments using miR-98-resistant PGRMC stably expressing ESCs to directly prove the implication of the miR-mediated PGRMC1 regulation in decidualization. We are transfecting the miR-resistant PGRMC1 expression vector into ESCs cell lines to explore the role of PGRMC1 in decidualization but is not going well. As you advised, lentiviral transduction of the PGRMC1 along with fluorescent protein may enable us to effectively select the overexpressing cells without losing differentiation status in response to decidual stimuli. We have acknowledged this and suggested it as a topic for further research in the “Limitation” section of the revised manuscript following editor’s comment (page 10, lines 336-348). “Limitations: In this study, we proposed that miR-98-mediated PGRMC1 downregulation may be involved in decidualization, but were unable to examine the effects of miR-98-resistant PGRMC1 on differentiation due to technical difficulties establishing PGRMC1-overexpressing ESCs. miR-98 may not be the only miRNA that modulates PGRMC1 expression; therefore, comprehensive analysis of transcriptional, post-transcriptional, and post-translational regulation of endometrial PGRMC1 during decidualization is required. In addition, PGRMC1 can bind to various proteins, including receptors for epidermal growth factor [46] and insulin [47], cytochrome P450 [48], and serpine mRNA-binding protein 1 (SERBP1) [19]. Further, PGRMC1 may interact with proteins associated with endomembrane trafficking/cytoskeleton and mitochondrial functions in decidualizing ESCs [49]. Thus, we plan to investigate the modulation of endometrial PGRMC1 expression and the interaction between PGRMC1 and intracellular proteins during decidualization in the future.”
In response the authors have made minimal changes to the manuscript, effectively restricted to rewording the title, and several minor text changes. Unfortunately, the authors have been unable to demonstrate the direct involvement of PGRMC1 downregulation in decidualization. Since this is the main publishable finding, the results as they stand do not merit publication. The authors mention technical difficulties in establishing ESCs that express miRNA-resistant PGRMC1. This may be due to stable plasmid selection conditions, which may affect the differentiation status of the ESC cells. It would be possible to introduce an expression cassette via a lentiviral vector at MOI 1. An inducible vector may be advisable, such as tetracycline, IPTG, or other available systems. Such vectors typically include fluorescent protein reporters, enabling discrimination between infected and non-infected cells in one plate, or population separation by FACS.