Role of ferroptosis in pregnancy related diseases and its therapeutic potential

Ferroptosis is a form of regulated cell death characterized by iron overload, overwhelming lipid peroxidation, and disruption of antioxidant systems. Emerging evidence suggests that ferroptosis is associated with pregnancy related diseases, such as spontaneous abortion, pre-eclampsia, gestational diabetes mellitus, intrahepatic cholestasis of pregnancy, and spontaneous preterm birth. According to these findings, inhibiting ferroptosis might be a potential option to treat pregnancy related diseases. This review summarizes the mechanisms and advances of ferroptosis, the pathogenic role of ferroptosis in pregnancy related diseases and the potential medicines for its treatment.

psoriasis  and rheumatoid arthritis (Long et al., 2022), Serving as the maternal-fetal interface, placenta plays a central role in maternal and fetal health during pregnancy. Placenta insufficiency, however, is tightly associated with pregnancy related diseases (PRDs), such as pre-eclampsia, gestational diabetes mellitus (GDM), and intrahepatic cholestasis of pregnancy (ICP) (Cindrova-Davies and Sferruzzi-Perri, 2022). Recently, there has been a growing appreciation for the importance of ferroptosis in PRDs. In this review, we summarized the molecular mechanisms of ferroptosis, as well as its pathogenic role and potential medicines in PRDs (Figure 1; Figure 2).

Iron in ferroptosis
Iron overload is a hallmark of ferroptosis. Iron drives ferroptosis mainly by two ways. Iron may directly generate excessive lipid reactive oxygen species (ROS) through the Fenton reaction (Conrad and Pratt, 2019). What's more, Fe 2+ acts as a cofactor of LOXs or prolyl hydroxylase (Doll and Conrad, 2017), which are enzymes responsible for lipid peroxidation and oxygen homeostasis (Kagan et al., 2017). Consequently, Fe 2+ promotes the production of lipid ROS and contributes to ferroptosis indirectly. Therefore, iron metabolism, including iron uptake, transportation, utilization, may affect cell susceptibility to ferroptosis. Firstly, Fe 3+ imports by binding to transferrin (TF), which can be recognized by transferrin receptor-1 (TfR1) in the cell membrane. And then, Fe 3+ endocytosis in endosomes, where it is reduced to Fe 2+ by six-transmembrane epithelial antigens of the prostate 3 (STEAP3). Finally, Fe 2+ is transported to the cytosolic labile iron pool via divalent metal transporter 1 (DMT1) (El Hout et al., 2018). Fe 2+ also comes from hemin and hemoglobin via the lysis of red blood cells, leading to ferroptosis (Kwon et al., 2015). It can be used in cellular processes or stored into ferritin, consisting of ferritin light chain (FTL) and ferritin heavy chain 1 (FTH1) (Ryu et al., 2017). But ferritin can be degraded by lysosomes through nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy (Hou et al., 2016). Ferroportin (FPN1) is responsible for exporting iron (Donovan et al., 2000), resisting to ferroptosis. PE, pre-eclampsia: gestational hypertension with proteinuria > 0.3g/L/day in the absence of a urinary tract infection or the abrupt onset of hypertension and proteinuria after 20 weeks of gestation (ACOG Practice Bulletin, 2019). GDM, gestational diabetes mellitus: diabetes first diagnosed in the second or third trimester of pregnancy that is not clearly either preexisting type 1 or type 2 diabetes (American Diabetes Association, 2018). ICP, intrahepatic cholestasis of pregnancy: characterized by maternal pruritus and increased serum bile acid concentrations, typically resolving postpartum (Chappell et al., 2019). SA, spontaneous abortion: pregnancy loss at less than 20 weeks' gestation in the absence of elective medical or surgical measures to terminate the pregnancy (Wilcox et al., 1988). abrAbbreviations: AA, arachidonic acid; AdA, adrenic acid; PRDs, pregnancy related diseases; GPX4, Glutathione peroxidase 4; GSH, glutathione; LOX, lipoxygenase; PUFA, polyunsaturated fatty acid; SD, sprague-dawley.
Frontiers in Cell and Developmental Biology frontiersin.org

Ferroptosis inhibitors
Ferroptosis is associated with a great number of diseases. Multiple genes and signaling pathways, associated with lipid and iron metabolism, and antioxidant systems, play a role in inhibiting ferroptosis, providing new potential therapeutic drugs for these diseases (Table 1).

Antioxidant systems
Antioxidant systems protecting cell from oxidative damage in ferroptosis are associated with multiple enzymes and proteins, including SLC7A11-GSH-GPX4 axis, CoQ10 system (Kuang et al., 2020).

Role of ferroptosis in pregnancy related diseases
Recently, basic research on ferroptosis in PRDs has gradually increased. Studies have indicated that placenta is susceptible to ferroptosis. Primarily, lipid peroxidation is frequent in placental injury (Schoots et al., 2018); Secondly, trophoblasts are abundant of iron: syncytiotrophoblasts extraordinarily highly expressed TfR1 (Seligman et al., 1979). Furthermore, Zrt-and Irt-like protein 8 (ZIP8) and Zrt-and Irt-like protein 14 (ZIP14), both of which play a roles in exporting iron from placental endosomal into the cytosol, are found at high levels in human placenta (Jenkitkasemwong et al., 2012). Three reviews described details on the role of iron and ferroptosis in the placenta (Ng et al., 2019;Beharier et al., 2021;Zaugg et al., 2022). Finally, decreased GPX4 levels have been associated with human placental dysfunction . Therefore, fully understanding the role of ferroptosis in placenta dysfunction may provide new treatment options for PRDs, including spontaneous abortion, PE, GDM, ICP, and spontaneous preterm birth (Figure 2).

Pre-eclampsia 4.1.1 Lipid peroxidation and pre-eclampsia
PE plays a leading role in maternal morbidity and mortality (Leitao et al., 2022). Ferroptosis has been related to the pathogenesis of PE . There are mounting evidence suggesting lipid peroxidation, is a major contributor for the damage of PE. A single-cell transcriptomics of the human placenta analysis indicated LPCAT3 and Sat1 (spermidine/ spermine N1-acetyltransferase 1) highly expressed in trophoblasts (Ng et al., 2019), both of which are related to ferroptosis (Dixon et al., 2015;Ou et al., 2016). Irwinda, R., et al. reported that the level of PUFAs significantly increased in PE patients (Irwinda et al., 2021;Liao et al., 2022). Furthermore, in rats model of PE, the concentration of MDA, the end product of lipid peroxidation, in the placenta has increased dramatically . Similarly, the levels of MDA in plasma and placenta are significantly elevated in PE patients (Aydin et al., 2004). To summarize, these studies suggest that lipid peroxidation leading to ferroptosis could contribute to PE.

Iron and pre-eclampsia
Iron overload is also associated with PE. Researchers have confirmed the concentration of plasma iron is higher in PE pregnancy than that in normal pregnancy (Liu et al., 2019). Yang et al., 2022a. reported the differentially expressed ferroptosis-related genes (FRGs) in early-onset PE were mainly enriched in iron-related pathways, including FTH1, FTL. Importantly, iron is abundant in trophoblasts under physiological conditions or in the context of iron deficiency (Sangkhae et al., 2020). What's more, the expression of FPN1 of trophoblasts decreased under hypoxic conditions , leading to the intracellular accumulation of Fe 2+ . Consequently, trophoblasts are vulnerable to ferroptosis. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, decreased the mortality rate of trophoblasts (Beharier et al., 2020). Similarly, the ferroptosis inhibitor improved the PE symptoms in a rat model, with the reduction of MDA . Thus, reducing the concentration of Fe 2+ might be a good way for PE treatment.

The SLC7A11-GSH-GPX4 axis and preeclampsia
Disorder of antioxidant system mediates ferroptosis in PE. A microarray analysis identified that miRNA-30b-5pm, which is in charge of reducing the expression of SLC7A11, upregulated in PE placental tissues. Also, they found SLC7A11 and GPX4 were decreased in PE placental tissues via GSE10588 data set . In accordance with other studies, the levels of SLC7A11, GSH and GPX4 declined while MDA levels were significantly increased (El-Khalik et al., 2022;D'Souza et al., 2016), indicating ferroptosis is involved in the pathogenesis of PE through the SLC7A11-GSH-GPX4 axis. A genomewide methylome analysis found the expression of ATF3, suppressing the system Xc− by binding to the SLC7A11 promoter , is higher in PE placenta than the normal placenta (Ching et al., 2014). As a result, human trophoblasts are susceptible to ferroptosis by the depletion or inhibition of GPX4 (Kajiwara et al., 2022). Additionally, pannexin 1 (Panx1) and toll-like receptor 4 (TLR4), which had a negative correlation with SLC7A11, are demonstrated to induce ferroptosis in PE (El-Khalik et al., 2022). Conversely, anti-ferroptosis factors can protect trophoblasts against ferroptosis through the SLC7A11-GSH-GPX4 axis. The level of Nrf2, which is responsible for promoting transcriptions of SLC7A11 and GPX4 , is lower in PE rats (Ju et al., 2022). DJ-1 plays a Frontiers in Cell and Developmental Biology frontiersin.org protective role in the process of ferroptosis in PE via the Nrf2/ GPX4 signaling pathway (Liao et al., 2022). These studies demonstrated that the SLC7A11-GSH-GPX4 axis plays a role in the pathogenesis of PE.

Gestational diabetes mellitus 4.2.1 Lipid peroxidation and gestational diabetes mellitus
Gestational diabetes mellitus (GDM) is common during pregnancy and is increasing in prevalence globally (Sweeting et al., 2022). The incidence of GDM ranges from 6.6% to 45.3% of pregnancies (Brown and Wyckoff, 2017) and one in six live births worldwide were complicated by GDM (Atlas, 2015). GDM is associated with long-lasting complications in the short and long term, such as macrosomia , dystocia (Crowther et al., 2022), childhood obesity in the child (Choi et al., 2022), recurrence of GDM (Giuliani et al., 2022), developing type 2 diabetes (Vounzoulaki et al., 2020) and cardiovascular disease in the mother (Christensen et al., 2022). Emerging evidence suggests ferroptosis contributes to the pathogenesis of GDM Gautam et al., 2021;Zhang et al., 2022c;Hu et al., 2022;Zaugg et al., 2022). The insulin sensitivity shifts depending on the requirements of pregnancy, which is an important metabolic adaptation during healthy pregnancy (Di Cianni et al., 2003). However, excessive insulin resistance in GDM promotes endogenous glucose production and the breakdown of fat stores, increasing the levels of blood glucose and free fatty acid (FFA) (Phelps et al., 1981). Indeed, glucose metabolism disorder is often accompanied by lipid metabolism disorder in GDM (Parhofer, 2015). A study indicated that women with GDM had significantly higher triglyceride (TG) concentrations (Hu et al., 2021b). In vitro model, the death rate of trophoblasts significantly increased after the co-treatment of high lipid (HL) and high glucose (HG). Furthermore, it was found that HL and HG can induce GDM in pregnant rats, leading to the damage of rats' placenta (He et al., 2021). The expression of ACSL4 significantly increased in placental tissues, as well . Consequently, excessive FFA of GDM may cause an increase in the level of lipid peroxidation, resulting in ferroptosis.

Iron and gestational diabetes mellitus
Iron overload, leading to oxidative stress damage, could promote the pathogenesis of GDM (Gautam et al., 2021;Zhang et al., 2022c;Zaugg et al., 2022). As reported, both elevated plasma ferritin concentrations and iron supplementation in pregnant women having adequate iron stores are risk factors of GDM . In GDM vivo model, the levels of iron deposition significantly increased , inducing the production of ROS via the Fenton reaction. As a result, oxidative damage leads to the injury and ferroptosis of pancreatic β-cell in GDM (Gautam et al., 2021;Du et al., 2022). The SLC7A11-GSH-GPX4 axis also contributes to GDM. The serum lipid peroxidation was higher, while the serum GPX4 concentration was lower in GDM women (Mauri et al., 2021). In summary, mounting evidence may suggest that excessive iron, and reduced GPX4 levels, two hallmarks of ferroptosis, are associated with GDM. However, experiments testing this hypothesis are still lacking.

Intrahepatic cholestasis of pregnancy
Intrahepatic cholestasis of pregnancy (ICP) is a complication, most occurs in the third trimester, in 0.3%-15% of pregnancies in various populations (Wikström Shemer et al., 2013). It is characterized by pruritus, elevated serum bile acid levels and liver transaminases, leading to meconium-stained amniotic fluid, fetal distress, preterm birth, and stillbirth (Wikström Shemer et al., 2013). There is increasing evidence that oxidative stress induced by bile acids leads to the pathogenesis of ICP (Sanhal et al., 2018). ICP patients had significantly lower levels of Se and GPX4 than normal pregnancies (Reyes et al., 2000;Hu et al., 2015). Moreover, patients with ICP had significantly higher level of MDA (Zhu et al., 2019). Analysis of differentially expressed ferroptosis-related genes in ICP and healthy pregnant showed EGFR, mediating ferroptosis, was higher upregulated in human placenta (Fang and Fang, 2022). Modification of oxidative stress caused by ferroptosis might be a treatment target for ICP. Further research, particularly in vivo and in vitro experiments, is needed to characterize the association between ferroptosis and ICP.

Other pregnancy-related disease
Excessive ferroptosis occurred in spontaneous abortion rat model with low levels of GSH, GPX4 and increased levels of TFR1, ACSL4 and MDA (Meihe et al., 2021). Some evidence also indicated spontaneous preterm birth is related to ferroptosis (Beharier et al., 2020). But few studies reported that the exact mechanism of ferroptosis and spontaneous abortion and spontaneous preterm birth are still unclear.

Potential medicines for ferroptosis in pregnancy related diseases
Trophoblast ferroptosis may provide a useful therapeutic target for pregnancy-related diseases. Quercetin, as an antioxidant, can significantly promote trophoblast invasion during early pregnancy via significantly increasing GSH levels (Ebegboni et al., 2019). Additionally, quercetin has positive effects on pre-eclampsia rats induced by L-NAME (Yang et al., 2019a;Yang et al., 2022b). Iron chelators, deferoxamine and ferrostatin-1, were indicated to decrease the concentration of placenta MDA in the PE rat mode, thereby blocking trophoblast ferroptosis (Beharier et al., 2020;Zhang et al., 2020). Similarly, vitamin E plays a role in the preventing PE by mitigating lipid peroxidation in placenta (Raijmakers et al., 2004). Thiazolidinediones, inhibiting ACSL4 against ferroptosis, is also oral antidiabetic drug by sensitizing tissue to the effects of insulin (Pollex and Hutson, 2011). A recently published case series demonstrate thiazolidinediones is safe during pregnancy (Haddad et al., 2008), indicating its potential therapeutic role for GDM. Trophoblasts ferroptosis may contribute to ICP, while the low concentration of Se is related to the pathogenesis of ICP (Reyes et al., 2000). Thus, Se, upregulating the expression of GPX4, can protect placental trophoblasts against oxidative stress, particularly ICP (Habibi et al., 2021). CoQ10 is significantly decreased in patients with ICP (Martinefski et al., 2014). Furthermore, CoQ10 supplementation improves estradiol-induced Frontiers in Cell and Developmental Biology frontiersin.org cholestasis in rats. CoQ10 supplementation is very well tolerated and has no clinically relevant toxic side effects in humans (Hidaka et al., 2008;Martinefski et al., 2020). Therefore, it would be an alternative therapy for women with ICP. Lack of 1,25(OH) 2 D 3 is related to PRDs, which may result from ferroptosis, such as spontaneous abortion, GDM and PE (Bespalova et al., 2019;de Souza and Pisani, 2020). Vitamin D elevated the level of GSH, GPX4 and reduced MDA through activation of the Nrf2/HO-1 pathway to suppresses ferroptosis. Therefore, vitamin D supplementation may be a strategy to improve PRDs. Previous studies reported astaxanthin significantly reduced the content of MDA in preeclamptic rats and trophoblast cell line (Xuan et al., 2014;Xuan et al., 2016;Fu et al., 2021). Certainly, drug efficacy and safety are quite important for pregnant woman and fetus. Further research may shed light on potential targeting drugs for ferroptosis in PRDs.

Conclusions and perspectives
Ferroptosis is a form of regulated cell death involving lipid metabolism, iron metabolism and antioxidant system, regulated by multiple genes and signaling pathways. PRDs are mainly associated with placenta dysfunction due to trophoblasts injury and death. Recently, an increasing number of experimental studies are exploring role of ferroptosis in PRDs in order to provide new potential therapeutic drugs and therapeutic targets for it. However, there are numerous problems that have not been elucidated on the association between ferroptosis and pregnancy related diseases. Firstly, the exact molecular mechanism of transplacental iron transport is not clear, though much work has been done on it. Secondly, ferroptosis is a form of cell death that is associated with lots of signaling pathways, like hypoxia signaling (Zou et al., 2019), AMP-activated protein kinase signaling (Li et al., 2020), E-cadherin-NF2-Hippo-YAP pathway (Yang et al., 2019b), and NRF2-KEAP1 pathway (Anandhan et al., 2020). However, the regulation of ferroptosis in placenta also remains a pressing challenge. Finally, we still do not know whether ferroptosis of trophoblasts leads to PRDs, or it is the execution pathway of PRDs. Therefore, extensive investigation is needed to explore it.

Author contributions
JX and CM conceived and designed the work. JX, FZ, XW, and CM wrote and revised the manuscript. All authors contributed to the article, read, and approved the final manuscript.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.