Simultaneous Ablation of Uterine Natural Killer Cells and Uterine Mast Cells in Mice Leads to Poor Vascularization and Abnormal Doppler Measurements That Compromise Fetal Well-being

Abstract

Intrauterine growth restriction (IUGR) is a serious pregnancy complication with short- and long-term health consequences. The mechanisms underlying this condition are not well understood. Animal models are the basis for understanding the causes of IUGR and for developing useful therapeutic strategies. Here, we aimed to ascertain the in utero growth of fetuses from NK (natural killer cells)/MC (mast cells)-deficient mothers that give birth to growth-restricted pups and to determine the time point at which IUGR starts. We used high frequency ultrasound imaging to follow-up fetal and placenta size and employed Doppler measurements to document blood supply to the fetus in females that were deficient for NK cells and MCs. In mice lacking NKs and MCs, we observed significantly reduced implantation sizes from mid gestation onward, which was further associated with smaller placentas. Additionally, NK/MC-deficiency was associated with absent and reversed end diastolic flow in umbilical arteries of the fetuses and an increased systolic/diastolic ratio as well as an elevated resistance index. Together, our results indicate that NKs/MCs promote blood flow, placental growth, and subsequent fetal development. The results of this study offer new insights as to how fetal growth is affected in vivo in NK/MC-deficient mice, whose pups are growth restricted at birth. The use of IUGR models and modern technologies enabling the in vivo follow-up of fetal development are important tools for understanding mechanisms behind pregnancy complications that in the future may lead to the development of effective therapies.

Keywords: Doppler measurements; angiogenesis; fetal development; intrauterine growth restriction; mast cells; natural killer cells; placenta; ultrasound imaging.

Figure 1

Comparison of implantation areas at gd5, 8, 10, and 12. (A) Implantation areas from WT Cpa3^+/+ + PBS mice (mice n = 2–5, implantations n = 6–31 per day) and MC/NK-deficient Cpa3^Cre/+ + anti-CD122 mice (mice n = 3, implantations n = 8–16 per day) at gd5, 8, 10, and 12. Results are presented as individual values for each single implantation and mean. Statistical differences were obtained using unpaired t-test (**P < 0.01, ***P < 0.001). (B) Representative ultrasound images from Cpa3^+/+ + PBS mice at gd5 (i), gd8 (ii), gd10 (iii), and gd12 (iv). gd, gestation day; WT, wild type; MC, mast cell; NK, natural killer cell.

Figure 2

Placental measurements at gd10, 12, and 14. (A) Representative ultrasound image of a WT implantation at gd10 showing the decidua basalis, the placenta, and the embryo. (B) Representative ultrasound image of a WT implantation at gd12 showing placental thickness (thick) and placental diameter (dia). Placental area (C), placental thickness (D), and placental diameter (E) from WT Cpa3^+/+ + PBS mice (mice n = 3–5, placentas n = 12–22 per day) and MC/NK-deficient Cpa3^Cre/+ + anti-CD122 mice (mice n = 3–4, placentas n = 8–14 per day) at gd10, 12, and 14. Results are presented as individual values for each single placenta and mean. Statistical differences were obtained using unpaired t-test (*P < 0.05, ** P < 0.01). gd, gestation day; WT, wild type; thick, thickness; dia, diameter; MC, mast cell; NK, natural killer cell.

Figure 3

Fetal and placental weight measurements and feto-placental index (FPI) at gd14. Fetal weights (A), placental weights (B), and FPIs (C) from progeny of WT Cpa3^+/+ + PBS mice (mice n = 4, fetus/placentas n = 35) and MC/NK-deficient Cpa3^Cre/+ + anti-CD122 mice (mice n = 3, fetus/placentas n = 28) at gd14. Results are presented as individual values and mean. Statistical differences were obtained using unpaired t-test (*P < 0.05, ** P < 0.01). gd, gestation day; WT, wild type; MC, mast cell; NK, natural killer cell.

Figure 4

Analysis of uterine artery velocities at gd10. (A) Representative Pulse-wave Doppler images from WT Cpa3^+/+ + PBS mice showing PSV and EDV. PSV (B), EDV (C), and resistance index (D) of uterine arteries from Cpa3^+/+ + PBS (n = 3) and Cpa3^Cre/+ + anti-CD122 (n = 3) mice at gd10 of pregnancy. Data are presented as mean with SEM. Statistical analysis was performed using the Mann–Whitney U test. gd, gestation day; WT, wild type; MC, mast cell; NK, natural killer cell; PSV, peak systolic velocity; EDV, end diastolic velocity.

Figure 5

Analysis of umbilical artery velocities at gd14. (A) Representative Color Doppler image of a fetal UmA at gd 14. (B) Representative Pulse-wave Doppler images from Cpa3^+/+ + PBS (i) and Cpa3^Cre/+ + anti-CD122 (ii, iii) mice, showing normal end diastolic flow (i), absent end diastolic flow (ii), or reversed end diastolic flow (iii). PSV (C), EDV (D), systolic/diastolic ratio (E), and resistance index (F) of UmAs of fetuses from Cpa3^+/+ + PBS (mice n = 3, UmA measurements n = 7) and Cpa3^Cre/+ + anti-CD122 (mice n = 3, UmA measurements n = 10) mice at gd14. Data are presented as mean with SEM. Statistical analysis was performed using the unpaired t-test (*P < 0.05). UmA, umbilical artery; gd, gestation day; PSV, peak systolic velocity; EDV, end diastolic velocity.