(126) Uncovering the Role of SLC40A1 and Ferroptosis in Endometriosis

Rupak Thapa^a,b, Suni Tang^a,b, Dominique I. Cope^a,b, Peixin Jiang^a, Fei Yuan^a,b, Linda Alpuing Radilla^c, Xiaoming Guan^c, Diana Monsivais^a,b

^aDepartment of Pathology & Immunology; ^bCenter for Drug Discovery; ^cDepartment of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, 77030, USA.

Abstract Text:

Endometriosis, affecting approximately 10% of women of reproductive age, is linked to infertility and pregnancy complications, with dysregulated iron homeostasis and oxidative stress contributing to these issues. In endometriosis, alterations in SLC40A1 expression may disrupt iron regulation in endometrial tissue, leading to oxidative stress and ROS production, which impairs decidualization and affects early pregnancy outcomes. We hypothesize that targeting iron regulation may offer a potential therapeutic strategy for improving reproductive health in women with endometriosis. We utilized human endometrial biopsies and mice with conditional Slc40a1 knockout (using progesterone receptor cre, “Slc40a1 cKO”) to explore the role of SLC40A1 in fertility. In a six-month fertility trial, Slc40a1 cKO females exhibited significant subfertility (7.25 ± 2.29 in controls vs 3.72 ± 3.3 pups/female in cKO, n=5 per group).  A superovulation study revealed no statistical difference in the average number of oocytes (34.5 ± 4.17, in controls vs. 51.83 ± 8.23, n=5 per group) in Slc40a1 cKO mice and no significant differences in estrus cycles, suggesting normal ovarian function. In timed mating analyses, Slc40a1 cKO mice showed a reduced number of implantation sites (8.5 ± 0.29 in controls vs. 5.5 ± 0.78 in Slc40a1 cKO) on day 6.5 post-coitus. Compared to controls, body, and uterine weights were significantly lower in Slc40a1 cKOs after the fertility trial, although ovarian weights remained unchanged (n=5 per group). In vitro, primary endometrial cells from endometriosis patients induced to artificially decidualize with estrogen, progesterone and cyclic AMP treatment showed reduced relative SLC40A1 expression (both mRNA and protein levels) compared to normal controls. RNA sequencing analysis revealed that SLC40A1 knockdown using siRNA led to a marked decrease in the expression of antioxidant defense genes (e.g., NQO1, SOD2, ADH1B, GPX4, HMOX1, SOD3, NFE2L1, PRDX6), while inflammation-related genes (e.g., TNFSF15, CTGF, TNFSF18, TNFSF4, MMP12, CD274) were significantly upregulated. The ferroptosis-related analysis identified the upregulation of ferroptosis driver genes (e.g., GRIA3, TGFBR1) and downregulation of ferroptosis suppressors (e.g., PRKX6, ENPP2, PIR, SLC40A1). Analysis of oxidative stress with Cell ROX revealed that SLC40A1 knockdown increased reactive oxygen species compared to cells treated with non-targeting siRNA. We identified that the SLC40A1 promoter contains FOXO1 and GATA2 binding sites, suggesting that these are key transcription factors potentially regulating SLC40A1 expression. Overall, our findings suggest that SLC40A1 dysregulation contributes to impaired uterine function in endometriosis by promoting oxidative stress and ferroptosis.

Keywords: Endometriosis, SLC40A1, oxidative stress, ferroptosis.

These studies were supported by grants from the R01 HD105800, T32 HD098068-05, and the Next Gen Pregnancy Initiative from the Burroughs Welcome Fund.