Iron-dependent apoptosis causes embryotoxicity in inflamed and obese pregnancy

Iron is essential for a healthy pregnancy, and iron supplementation is nearly universally recommended, regardless of maternal iron status. A signal of potential harm is the U-shaped association between maternal ferritin, a marker of iron stores, and risk of adverse pregnancy outcomes. However, ferritin is also induced by inflammation and may overestimate iron stores during inflammation or infection. In this study, we use mouse models to determine whether maternal iron loading, inflammation, or their interaction cause poor pregnancy outcomes. Only maternal exposure to both iron excess and inflammation, but not either condition alone, causes embryo malformations and demise. Maternal iron excess potentiates embryo injury during both LPS-induced acute inflammation and obesity-induced chronic mild inflammation. The adverse interaction depends on TNFα signaling, causes apoptosis of placental and embryo endothelium, and is prevented by anti-TNFα or antioxidant treatment. Our findings raise important questions about the safety of indiscriminate iron supplementation during pregnancy.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. For the initial experiments described in Figure 1A-E, we performed sample-size calculation by Fisher exact test. We assumed that after LPS injection, the rate of complications is 20% in non-iron-loaded pregnancies vs 100% in high-iron pregnancies, 6 dams/group revealed significant differences in outcome with power of 0.9 and p< 0.05. For all subsequent mouse experiments, sample sizes were decided based on the initial calculations and expected outcomes. Sample sizes for in vivo experiments are indicated in each figure panel. For in vitro experiments, at least 3 independent experiments were performed in order to calculate statistical significance and verify results. The number of experiments performed for in vitro data are indicated in each figure panel.
We did not exclude any data from analyses.
The number of replicates for each experiment are indicated in each figure panel. All attempts of replication were successful.
Mice were randomly allocated into the experimental groups. Because each pregnant mouse can have up to 12 fetuses, not all placentas/fetuses were analyzed from all pregnancies. Rather, placentas/fetal tissues were randomly selected for analyses so that multiple samples were analyzed from at least 3 or more different pregnancies. For in vitro studies, randomization was not applicable to our experimental set up.
Investigators were not blinded during the study because the investigators who generated mouse samples also analyzed those samples.

April 2020
Validation Anti-mouse IgG HRP antibody 7076, Cell Signaling Technology, 1:5,000 Anti-rabbit IgG HRP antibody 7074, Cell Signaling Technology, 1:5,000 Anti-goat IgG HRP antibody 2354, Santa Cruz, 1:5,000 ImmPRESS horse anti-rabbit IgG HRP detection kit MP-7401, Vector Laboratories ImmPRESS horse anti-goat IgG AP detection kit MP-5405, Vector Laboratories Neutralizing antibodies: Human TNF! neutralizing Rabbit monoclonal antibody 7321, Cell Signaling Technology Mouse TNF! neutralizing Rat monoclonal antibody clone XT3.11, BioXcell Rat IgG isotype control, anti-trinitrophenol Rat monoclonal antibody clone TNP6A7, BioXcell All the antibodies used in the study were validated by the manufacturing companies for mouse and human specificity and for the applications described in the study. All antibodies were further validated by our laboratory via expected and predicted molecular weight by Western blotting and including controls that are known to increase or decrease expression of the proteins visualized. For example, TNF treatment is known to induce cleaved caspase-3, and iron is known to induce expression of ferritin. For all antibodies reported, optimal dilutions were determined by our laboratory for the indicated applications.
Antibody validation by the manufacturing companies are as follows: 1. Mouse and human cleaved caspase-3 Rabbit monoclonal antibody 9664, clone 5A1E, Cell Signaling Technology: Cleaved Caspase-3 Rabbit mAb detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. This antibody does not recognize full length caspase-3 or other cleaved caspases. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to amino-terminal residues adjacent to Asp175 of human caspase-3, and reacts with human, mouse, rat, and monkey samples which was determined by testing in at least one approved application (see https://www.cellsignal.com/datasheet.jsp?productId=9664&images=1&protocol=0) 2. Mouse and human total caspase-3 Rabbit polyclonal antibody 9662, Cell Signaling Technology: Caspase-3 Antibody detects endogenous levels of full-length caspase-3 (35 kDa). Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the cleavage site of human caspase-3. Antibodies are purified by protein A and peptide affinity chromatography. Antibody reacts with human, mouse, rat, and monkey samples which was determined by testing in at least one approved application (see https://www.cellsignal.com/datasheet.jsp?productId=9662&images=1&protocol=0) 3. Mouse and human ferritin heavy chain Rabbit monoclonal antibody 4393, clone D1D4, Cell Signaling Technology: FTH1 (D1D4) Rabbit mAb recognizes endogenous levels of total FTH1 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human FTH1 protein. Antibody reacts with human, mouse, rat, and monkey samples which was determined by testing in at least one approved application (see https://www.cellsignal.com/ datasheet.jsp?productId=4393&images=1&protocol=0).
6. Mouse cleaved caspase-1 Rabbit monoclonal antibody 89332, clone E2G2I, Cell Signaling Technology: Cleaved Caspase-1 (Asp296) (E2G2I) Rabbit mAb recognizes endogenous levels of caspase-1 protein only when cleaved at Asp296. A non-specific band is detected at 70 kDa in some cells. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp296 of mouse caspase-1 protein. Antibody reacts with mouse samples which was determined by testing in at least one approved application (see https://www.cellsignal.com/datasheet.jsp?productId=89332&images=1&protocol=0). 7. Human caspase-9 Mouse monoclonal antibody 9508, clone C9, Cell Signaling Technology: Caspase-9 (C9) Antibody detects endogenous levels of the pro form of caspase-9 as well as cleaved fragments. Monoclonal Antibody is produced by immunizing mice with a recombinant human caspase-9 protein. Antibody reacts with human, mouse, rat, hamster, and monkey samples which was determined by testing in at least one approved application (see https://www.cellsignal.com/datasheet.jsp? productId=9508&images=1&protocol=0).
8. Mouse and human electron transport chain Total OXPHOS Rodent WB Antibody Cocktail ab110413, Abcam: Total OXPHOS Rodent WB Antibody Cocktail ab110413 is an optimized cocktail of high-quality antibodies for analyzing relative levels of OXPHOS complexes in rat or mouse mitochondria by western blot. Antibody reacts with mouse, rat, cow, human, and cynomolgus monkey (see https:// www.abcam.com/total-oxphos-rodent-wb-antibody-cocktail-ab110413.pdf).