Binding of microRNA-135a (miR-135a) to homeobox protein A10 (HOXA10) mRNA in a high-progesterone environment modulates the embryonic implantation factors beta3-integrin (ITGβ3) and empty spiracles homeobox-2 (EMX2).

Original Article Page 1 of 12 Binding of microRNA-135a (miR-135a) to homeobox protein A10 (HOXA10) mRNA in a high-progesterone environment modulates the embryonic implantation factors beta3-integrin (ITGβ3) and empty spiracles homeobox-2 (EMX2) Xi Luo1,2,3,4#^, Renxiang Yang2#, Yun Bai3,4, Lei Li3,4, Na Lin3,4, Lan Sun2, Jianjun Liu5, Ze Wu3,4^ 1 Faculty of Life science and Technology, Kunming University of Science and Technology, Kunming, China; 2Medical School, Kunming University of Science and Technology, Kunming, China; 3Department of Reproductive Medicine, the First People’s Hospital of Yunnan Province, Kunming, China; 4Reproductive Medical Center of Yunnan Province, the Affiliated Hospital of Kunming University of Science and Technology, Kunming, China; 5Yunnan Key Laboratory of Stem Cell and Regenerative Medicine, Research Center of Biomedical Engineering, Kunming Medical University, Kunming, China Contributions: (I) Conception and design: X Luo, R Yang, Z Wu; (II) Administrative support: J Liu, Z Wu; (III) Provision of study materials or patients: Y Bai, L Li, N Lin; (IV) Collection and assembly of data: R Yang, L Sun; (V) Data analysis and interpretation: X Luo, J Liu, Z Wu; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. # These authors contributed equally to this work. Correspondence to: Ze Wu. Department of Reproductive Medicine, the First People’s Hospital of Yunnan Province, Kunming, China; Reproductive Medical Center of Yunnan Province, the Affiliated Hospital of Kunming University of Science and Technology, Kunming, China. Email: ; Jianjun Liu. Yunnan Key Laboratory of Stem Cell and Regenerative Medicine, Research Center of Biomedical Engineering, Kunming Medical University, Kunming, China. Email: . Background: Patients with elevated circulating progesterone concentrations on the day of the human chorionic gonadotropin (hCG) trigger had relatively low implantation rates during assisted reproductive treatments. In this study, we assess the hypothesis that different concentrations of progesterone regulate the expression of homeobox protein A10 (HOXA10) and its downstream genes through miRNA-135a. Methods: MicroRNA-135a (miR-135a), HOXA10, beta3-integrin (ITGβ3), and empty spiracles homeobox-2 (EMX2) expression levels in endometrial tissues from patients with elevated progesterone were measured. To determine the threshold of progesterone level which can impair implantation, Ishikawa cells were used to determine the expression of the aforementioned 4 genes after exposure to 5 graded concentrations of progesterone. The dual-luciferase reporter assay was used to verify whether miR-135a regulated the expression of HOXA10. Furthermore, the effects of HOXA10 on the expression of key endometrial receptivity genes ITGβ3 and EMX2 were confirmed. Results: High progesterone levels promoted miR-135a expression in vivo, and miR-135a bound to the 3'-untranslated region (3'-UTR) of HOXA10 mRNA to inhibit HOXA10 expression. Reduction of HOXA10 promoted EMX2 expression and inhibited ITG-3 production. Progesterone promoted the expression of HOXA10 in vitro at low concentrations. However, when the concentration was greater than 10−7 ng/mL, progesterone inhibited HOXA10 by promoting miR-135a expression, thereby altering the expression of related genes and affecting endometrial receptivity. Conclusions: In vitro, the trend in miR-135a expression (which first decreased and then increased) was in direct contrast to that of HOXA10 expression (which first increased and then decreased) as progesterone levels increased. The key factors regulating endometrial receptivity included ITGβ3 and EMX2, which were ^ ORCID: Xi Luo, 0000-0002-9779-3697; Ze Wu, 0000-0002-8899-0445. © Annals of Translational Medicine. All rights reserved. Ann Transl Med 2021;9(8):662 | http://dx.doi.org/10.21037/atm-21-596 Luo et al. High prog. modulates HOXA10, ITGβ3 and EMX2 by miR-135a Page 2 of 12 confirmed to be regulated by HOXA10. High progesterone levels affected miR-135a expression, and miR135a inhibited HOXA10 expression, thereby affecting endometrial receptivity. Keywords: Empty spiracles homeobox-2 (EMX2); homeobox protein A10 (HOXA10); implantation rate; beta3integrin (ITGβ3); microRNA-135a (miR-135a); progesterone elevation Submitted Dec 30, 2020. Accepted for publication Apr 01, 2021. doi: 10.21037/atm-21-596 View this article at: http://dx.doi.org/10.21037/atm-21-596 Introduction Since the advent of ART technology, more and more infertile couples have acquired their own children. In conventional ART treatment, the mature oocytes were retrieved for in vitro fertilization after control ovarian stimulation (COS), and embryos were transferred after 3–5 days of culture in vitro to obtain pregnancy. In the process of COS, it is necessary to monitor the female serum reproductive hormone levels, such as follicle stimulating hormone (FSH), estradiol (E2), luteinizing hormone (LH), progesterone, prolactin, and testosterone. Among them, the main role of progesterone is to protect the endometrium of women. During pregnancy, it can provide support and protection for the early growth and development of the fetus, and it can protect the fetus by inhibiting uterine contractions. As similar of LH in structure and function, exogenous human chorionic gonadotropin (hCG) is usually used to induce ovulation by causing artificial LH surge during ART treatment. Therefore, the main function of hCG trigger is to induce ovulation, and the serum progesterone level should be low on the day of hCG trigger. However, our previous retrospective study showed that patients with elevated progesterone levels on the day of the hCG trigger had poor outcomes with assisted reproductive technology (ART) treatment, despite non-significant differences in embryo quality among the 2,921 tested samples (1). In our more recent investigation, we further demonstrated that an elevation in plasma progesterone had differential effects on patients with different ovarian responses to hormonal stimulation (2). Hence, whole-embryo freezing and transfer of the thawed embryo are recommended for women with early elevated progesterone levels on the hCG trigger day. The intrinsic factors responsible for the ART outcome of increased progesterone on hCG trigger day has been a question we have explored. Existing studies have shown that progesterone plays a key role in embryonic implantation (3), especially in preparing the endometrium prior to implantation and regulating the invasion of trophoblast cells and their migration into the uterine wall. The major reason for poor ART treatment outcomes is that the increase in progesterone on hCG trigger day in the follicular phase can negatively affect embryonic implantation (4). Even if the embryo is of high quality, the implantation process cannot be completed without optimal endometrial conditions. A primary reason may be that the high concentration of progest (...truncated)