Advanced Maternal Age Impairs Uterine Artery Adaptations to Pregnancy in Rats

Figures

• 

  Figure 1. Pregnancy outcomes from young and aged dams at GD 20. (A) Litter size; (B) Reabsorptions; (C) Fetal crown-rump length (CRL):abdominal girth (AG) ratio; (D) Fetal weight; (E) Placental weight; (F) Fetal:placental weight ratio. Data presented as mean ± SEM; n represents litter average; n = 13–19 dams/group. Data are sex-specific means of litter outcomes, where applicable. Pregnancy outcomes were compared using a t-test (litter size), Mann Whitney U-test (reabsorptions), or a two-way ANOVA (dam age vs. fetal sex) followed by Sidak’s post hoc test. * p < 0.05; ** p < 0.01; **** p < 0.0001.

• 

  Figure 2. Active pressure myography outcomes for main uterine arteries from young (black) and aged (orange) nonpregnant (open circles) and pregnant (GD 20; closed circles) rats. (A) Percentage myogenic tone calculated from the intraluminal diameter (ID) measures using the equation: percentage myogenic tone = ((IDpassive − IDactive)/IDpassive) × 100, where IDactive is the intraluminal diameter when assessed in phosphate-buffered saline solution with calcium present to enable smooth muscle cell contraction, and IDpassive is the intraluminal diameter when assessed in a calcium-free solution containing papaverine to prevent smooth muscle contraction; (B) Percentage myogenic tone area under the curve (AUC), calculated from the entire pressure curve range of 4–160 mmHg; (C) The active pressure myography curve data, now presented as a percentage difference in ID from baseline (4 mmHg). Data presented as mean ± SEM; n = 7–10 rats/group. Percentage myogenic tone AUC was compared using a two-way ANOVA (age vs. pregnancy status) followed by Sidak’s post hoc test. ** p < 0.01; *** p < 0.001.

• 

  Figure 3. Passive (mechanical) pressure myography outcomes for main uterine arteries from young (black) and aged (orange) nonpregnant (open circles) and pregnant (GD 20; closed circles) rats. (A) Circumferential stress-strain; (B) Stress area under the curve (AUC); (C) Strain AUC; (D) Passive lumen diameter; (E) Passive lumen diameter AUC of arteries. All outcomes were assessed under calcium-free conditions to prevent smooth muscle cell contraction. Data presented as mean ± SEM; n = 10–24 rats/group. Outcomes were compared as AUC using a two-way ANOVA (age vs. pregnancy status) followed by Sidak’s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

• 

  Figure 4. Structure of main uterine arteries from young and aged nonpregnant and pregnant (GD 20) rats. Tunica intima and media (combined) percentage positive area for (A) collagen (Masson’s Trichrome; collagen is stained blue) and (B) elastin (Verhoff stain with Fast Green counterstain; elastin fibers are stained dark), and the (C) collagen:elastin ratio. Scale bar represents 100 µm. Data presented as mean ± SEM; n = 4–6/group. Outcomes were compared using a two-way ANOVA (age vs. pregnancy status) followed by Sidak’s post hoc test. * p < 0.05; ** p < 0.01.

• 

  Figure 5. Densitometry analysis of activity by MMP-2 from main uterine arteries of young (black) and aged (orange) nonpregnant (open circles) and pregnant (GD 20; closed circles) rats. Representative zymography gel: the first lane has a protein ladder. Both pro-MMP-2 (72 kDa) and active MMP-2 (62 kDa) are visible. However, only MMP-2 bands had a sufficiently detectable band able to be used for analysis. Densitometry assessment: bands of clearing represent gelatinase activity by MMPs. L, ladder; YNP, young nonpregnant; YP, young pregnant; ANP, aged nonpregnant; AP, aged pregnant. Data presented as mean ± SEM; n = 6/group. Outcomes were compared using a two-way ANOVA (age vs. pregnancy status) followed by Sidak’s post hoc test. * p < 0.05; ** p < 0.01.

• 

  Figure 6. Serum estradiol (E2) concentrations of young (black) and aged (orange) nonpregnant (open circles) and pregnant (GD 20; closed circles) rats. Data presented as mean ± SEM; n = 8/group. Outcomes were compared using a two-way ANOVA (age vs. pregnancy status) followed by Sidak’s post hoc test.