Vascular reactivity

Cleaned uterine arteries were cut into equal 2 mm rings and mounted on a wire myograph for measuring isometric tension (DMT 620M, Danish Myotechnology, Aarhus, Denmark) using stainless steel wires (25 μm). Each myograph chamber contained PSS kept at 37°C and pH 7.4 with constant administration of 95% O₂ and 5% CO₂. Blood vessels were stretched to their optimal tension during the normalization procedure. Each arterial ring was stretched in a stepwise manner. Wall tension and internal circumference at each stretch were calculated and plotted to provide a resting wall tension–internal circumference curve for that artery, using the DMT Normalization Module. Arterial rings were normalized to 0.9L ₁₀₀, where L ₁₀₀ is the internal circumference of the vessels at a transmural pressure of 100 mmHg. Optimal diameters (OD) were calculated as OD = 0.9L ₁₀₀/π (Yamaleyeva et al. 2015). After obtaining the OD vessel rings were allowed to equilibrate for 30 min before being challenged twice with 80 mM KCl (maximal response to KCl) and then rested in normal PSS again. After resting, constrictions to cumulative additions of phenylephrine (Ph, 10⁻⁹–10⁻⁵ M) were recorded. The responses to Ph were expressed as the percentage of maximal contraction induced by KCl. For endothelium‐dependent vasodilatation, uterine artery segments were preconstricted with a submaximal dose of Ph (1 μM) followed by cumulative additions of half‐log concentrations of acetylcholine (ACh, 10⁻⁹–10⁻⁵ M). To assess the contribution of nitric oxide (NO) to vasodilatation, arteries were incubated with the endothelial nitric oxide synthase inhibitor (eNOS) N ^ω‐nitro‐l‐arginine methyl ester hydrochloride (l‐NAME, 10⁻⁴ M) for 45 min before preconstruction with Ph and addition of ACh. Cumulative concentrations of sodium nitroprusside (SNP, 10⁻⁹–10⁻⁵ M) after preconstricion with Ph (1 μM) were used to assess endothelium‐independent relaxation.