*4.4. Placental Oxidative Stress*

The explants were treated with 2.3 mM xanthine (X) and 0.015 U/mL xanthine oxidase (XO) (Sigma-Aldrich) to induce oxidative stress [8,55]. Explants were incubated in X/XO in the presence or absence of 1 μg/mL hydroxychloroquine for 48 h at 37 ◦C in 20% oxygen, 5% CO2. Untreated cultures served as controls. Conditioned media were collected and stored at -80 ◦C in the presence of 0.005% butylated hydroxytoluene (BHT) (Sigma-Aldrich, St. Louis, Missouri, USA) to prevent autoxidation for activin A and 8-isoprostane assay measurements. Elevated levels of 8-isoprostane are a marker for lipid peroxidation caused by oxidative stress [56] and, in addition, high levels of activin A have been implicated in the pathway of placental oxidative stress [12].

#### *4.5. Measurement of sFlt-1, sEng, TNF-*<sup>α</sup>*, and Activin A with ELISA*

Levels of sFlt-1, sEng, TNF-<sup>α</sup>, and activin A were measured in placental explant (*n* = 10) conditioned media using Quantikine immunoassay ELISAs (R&D systems, Minneapolis, Minnesota, USA) according to the manufacturer's protocol. All samples were assayed in duplicate. Briefly, for the measurement of sFlt-1, sEng, TNF-<sup>α</sup>, and activin A, the conditioned media was diluted (1:40, 1:10, 1:5, and 1:30, respectively) with assay diluent. Results were normalized per milligram weight of tissue.

#### *4.6. Human Umbilical vein Endothelial Cell (HUVEC) Isolation*

Umbilical cords were also obtained from healthy women with term singleton pregnancies (*n* = 8) undergoing elective caesarean. HUVECs were isolated and cultured, as previously described, with minor modifications [8,57]. Briefly, the umbilical cord was severed from the placenta within an hour of collection. All areas with clamp marks were removed and the umbilical vein was cannulated and tied with thread. After removal of blood, the umbilical veins were infused with type II collagenase (0.5 mg/mL, Sigma-Aldrich) and incubated for 10 min at 37◦C to isolate the endothelial cells. They were maintained in M199 complete media containing 20% heat-inactivated fetal calf serum, 1% antibiotics/antimycotics (penicillin G, streptomycin sulphate, and amphotericin B), and 1% l-glutamine with endothelial and fibroblast growth factor (10 ng/mL each). Only cells at passage 2 to 4 were used for experiments.
