Effects of Different Oxygen Levels and Time Points on Mitochondrial Respiratory Functional Analysis of Placental Tissue

Sample Collection and Materials

This research recruitment period runs from December 25, 2020 to December 25, 2022. Placental tissues were collected from 50 normal term (37–40 weeks of gestation) pregnancies, delivered through elective Caesarean section (multiple Caesarean section history and cephalopelvic disproportion). Samples from the foetal side of the placenta (50 mg) were biopsied within 15 min (minute) of delivery, mixed with PBS (Phosphate Buffered Saline), and delivered on ice to the laboratory. Placental villous tissue samples were then prepared by referring to the processing as previously described in a video [13]. Simply put, placental tissue is placed in a 10 cm diameter petri dish containing PBS, thoroughly rinsed, and the blood is washed out to the greatest extent possible, then the left hand holds the tweezers, and the right hand holds the glass slide, scraping the villi tissue, and removing the fibrous tissue and blood vessels to the greatest extent possible.

Human Ethical Approval Statement

Subjects were recruited from the Qinghai University Affiliated Hospital with the approval of the Qinghai University Affiliated Hospital Ethics Committee, and after obtaining informed written consent.

High-Resolution Respirometry

Samples were then washed in ice-cold PBS and permeabilized in saponin (50 ug) in BIOPS (1 mL) solution for 40 min on ice with continual mixing. Samples were subsequently washed twice for 10 min on ice in MiR05 respiration medium, as has been previously described [14]. The speed of the shaker was approximately 100–110 r/min, although this speed could not be too fast in order to prevent the mitochondrial membrane from breaking. Next, blot-dried before measuring wet weight, 5–10 mg wet weight was added to the O2K chamber containing respiratory medium MiR05 at 37℃ and was then exposed to varying supplemental oxygen levels (150–160, 200, 250, and 300 μM). The machine used was located at an altitude of 2200 m above sea level in Xining, Qinghai Province, with an atmospheric pressure of 581.57 mmHg and an oxygen pressure of 16.24 kPa. The machine was also subjected to final and proper calibration using zeroing powder every three months. In addition, air calibration was performed before each experiment, with the oxygen concentration in the chamber after this being approximately 150–160 uM. The first part of this experiment was designed to verify the effect of different oxygen concentrations in the chamber on mitochondrial respiratory function, whilst the second part aimed to study the duration of osmotic mitochondrial activity stored in the mitochondrial respiratory fluid. Sample preparation is estimated to take 1.5 hours from delivery to penetration to detection of oxidative phosphorylation. It then took approximately 1.5 h further to measure a single parameter of mitochondrial respiratory function. Permeabilized placental villous tissue used for testing was first stored at 4 °C in a refrigerator. Here, mitochondrial viability was determined at 1.5~3, 3~4.5, 4.5~6, and 6~7.5 h from delivery. Substrate uncoupling inhibitor titration (SUIT) was then performed as previously described [14] (Figure1): For SUIT protocol pyruvate (5 mM), glutamate (10 mM) and malate (2 mM) were added to determine complex I (CI) mediated LEAK respiration, then oxidative phosphorylation (OXPHOS) through CI was stimulated by the addition of ADP（Adenosine Diphosphate） (5 mM). Cytochrome c (10 uM) was then added to test the integrity of the outer mitochondrial membrane. Succinate (10 mM) was added directly after cytochrome c, to elevate OXPHOS through complexes I and II (OXPHOS CI, II). Titrations of CCCP (0.5uM) were also used to investigate the ETS capacity with CI and CII (ETS CI, II). Rotenone (0.5uM) and antimycin A (2.5uM) were then added to inhibit Complexes I and III respectively, with N,N,N,N-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD:0.5 mM) and ascorbate (2 mM) being added to determine OXPHOS through complex IV.

Respiration of Placental Mitochondria under Different Oxygen Levels

Mitochondrial respiration capacity did not change significantly under the variation of oxygen levels. As shown in (Figure 2), mitochondrial respiration capacity in ambient air (150–160 μM) was not significant differently from when exposed to oxygen levels of 200, 250, and 300 μM in oxidative phosphorylation (OXPHOS) with substrates for Complex I (CI) and Complex II (CII). In addition, the same was observed for the maximum respiration of the electron transfer system (ETSmax). OXPHOS with substrates for Complex IV (CIV), non-phosphorylating respiration (LEAK), and non-electron transfer system respiration remained stable across different supplementary oxygen concentrations. These results indicated that changes in oxygen concentration in the O2K chamber used to detect the respiratory capacity of placental mitochondria did not alter mitochondrial respiratory function. This was sufficiently in line with the manufacturer’s recommendations and extensive literature demonstrating air calibration prior to closure of the system (a procedure that is required).

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However, this study was focused on mitochondrial metabolism in the highland adaptation to low oxygen levels. The equipment was used to conduct research at high altitudes 3780 m (meters) since high-resolution respirometry measurements cannot be performed on frozen materials. It was important to keep in mind that compared to low altitudes (below 2500 m), the machine calibration approach utilized here to study the characteristics of mitochondrial metabolism at high altitudes was different. The instrumental background could also be performed at the higher altitude [11, 15]. Altitude calibration schemes for different levels are different, and therefore close communication with engineers was required.

Oxygen was added to the tissue preparation prior to measuring respiratory function to ensure that oxygen did not limit respiratory capacity (especially at high respiratory rates) and to counteract the formation of oxygen diffusion gradients. When studying the effect of oxygen concentration on mitochondrial respiratory function, oxygen was injected into the chamber respiratory fluid via a syringe to limit the oxygen concentration in the chamber before the start of each experiment. Oxygen concentration in the chamber was also decreased with the addition of mitochondrial substrates, uncouplers, and inhibitors. It was found that despite the laboratory being located at an altitude of 2200 m, after O2K calibration, oxygen concentration in the chamber was 150– 160 uM. During the placental tissue experiment, by reducing the detected tissue to 5-10 mg, the experiment could be carried out smoothly without additional increases in oxygen concentration within the chamber. However, for heart and skeletal muscle tissue with high oxygen consumptions, it may be necessary to add enough oxygen to meet mitochondrial oxygen consumption.

Respiration of Placental Mitochondria in Different Time Points

The mitochondrial respiration capacity changed as time from placental delivery increased. As shown in (Figure 3), there was a significant decrease in OXPHOS(Oxidative phosphorylation) with substrates for mitochondrial CI, CI + CII, and ETSmax after 4.5 h storage in the respiratory medium compared with 3 h. OXPHOS with substrates for CIV, LEAK, and ROX did not significantly change at different time points. Despite being stored in respiratory medium, some activity of the placental mitochondria was impaired after 3h. Considering that it took 1.5 h (from delivery to penetration ready for testing) to prepare the tissue and 1.5 h (from the addition of tissue to the chamber to the final addition of the substrate of complex IV) to test a single mitochondrial respiratory parameter, we recommend testing no more than two parameters per sample so as not to exceed the 3 h time limit.

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Figure 3: Mitochondrial respiration capacity differs at different time points. Mitochondrial respiration through Complex I, Complex II, Complex IV, Leak Respiration, ETSmax, and ROX were measured at different time points after preparation (1.5~3, 3~4.5, 4.5~6, 6~7.5 h). Asterisk denotes significance between different time points. *P ≤ 0.05, P ≤ 0.01，*P ≤ 0.001, ****P ≤ 0.0001; ns, nonsignificant. Data are mean ± SE.

In conclusion, here we recommend not utilizing placental samples more than 3 h after delivery.