Objective
Our goal was to define mechanisms that protect murine pregnancies deficient in spiral arterial remodeling from hypertension, hypoxia, and intrauterine growth restriction.
Study Design
Microultrasound analyses were conducted on virgin, gestation day 2, 4, 7, 9, 10, 12, 14, 16, 18, and postpartum BALB/c (wild type) mice and BALB/c- Rag2 −/− /Il2rg −/− mice, an immunodeficient strain lacking spiral arterial remodeling.
Results
Rag2 −/− /Il2rg −/− dams had normal spiral arterial flow velocities, greatly elevated uterine artery flow velocities between gestational day 10-16 and smaller areas of placental flow from gestational day 14 to term than controls. Maternal heart weight and output increased transiently. Conceptus alterations included higher flow velocities in the umbilical-placental circulation that became normal before term and bradycardia persistent to term.
Conclusion
Transient changes in maternal heart weight and function accompanied by fetal circulatory changes successfully compensate for deficient spiral arterial modification in mice. Similar compensations may contribute to the elevated risk for cardiovascular diseases seen in women and their children who experience preeclamptic pregnancies.
Normal pregnancy significantly changes reproductive tract and systemic circulations to accommodate growth and metabolic demands of developing conceptuses. In species with hemochorial placentation, such as humans, uterine vascular changes include increased permeability, angiogenesis, and structural remodeling of most spiral arteries (SA) to reduce vasoactivity and increase capacity. In humans, inadequate modification of decidual and myometrial SA is often accompanied by preeclampsia (PE) and/or intrauterine growth restriction (IUGR). SA remodeling is progressive and involves loss of vascular smooth muscle coat and elastic lamina, mural invasion by trophoblast cells that deposit nonvasoactive fibrinoid and transient replacement of vascular endothelial cells by intravascular trophoblast. In humans, SA remodeling normally occurs during first and second trimester with approximately two-thirds of these vessels dilating 5-10 fold. SA remodeling slows maternal blood flow, minimizes turbulence, and optimizes exchange time with the fetal circulation. Decidual lymphocytes, mainly uterine natural killer (uNK) cells, contribute to early stages of decidual SA transformation through cytokine secretion as do mechanical flow properties such as shear forces and pressure.
The systemic circulatory changes of early normal pregnancy include increases in cardiac output, blood volume, and glomerular filtration rate that result in an overall maternal state of high blood flow with low vascular resistance. Inadequate or excessive cardiovascular adaptations before week 20 of pregnancy predict complications. Cardiac disease occurs in 1-4% of pregnancies in women with no preexisting heart abnormalities. Further, women who experience adverse outcomes such as PE, carry increased risks for cardiovascular and metabolic diseases into later life. Thus, identifying links between uterine vascular remodeling and systemic cardiovascular abnormalities is critical for appropriate management of human gestational complications. Early in normal human and mouse pregnancies, mean arterial blood pressure decreases slightly or is stable. This indicates that gains in uterine flow are more attributable to morphologic remodeling of uterine arteries than to alterations in their smooth muscle tone.
Mouse models have promoted understanding of mechanisms regulating gestational changes to the cardiovascular system. Comparisons of microultrasound measurements of placental growth and blood flow velocity between normal and mutant mice (ie, Nos3 −/− ) revealed significant similarities to human pregnancies. The reports on pregnancies in mutant mice examined by microultrasound do not include reports on animals without SA modification. Lack of SA modification is a characteristic of mice deficient in uNK cells. It is a phenotype readily reversed by transplantation of marrow from mice genetically deficient for T and B cell differentiation (ie, NK + T − B − marrow). SA modification is histologically detected between gestation day 9-10 of 19-20 day mouse pregnancy, a time-point recognized for allantoic fusion with the chorionic plate to open placental circulation. In this study, we examine hemodynamic features of wild-type BALB/c +/+ (WT; ie, NK + T + B + ) and alymphoid (deficient in NK, T, and B cells [NK − T − B − ]) BALB/c- Rag2 −/− /Il2rg −/− mice before, across, and after pregnancy using microultrasound. Unlike most patients with impaired SA remodeling, Rag2 −/− /Il2rg −/− dams with unmodified SA have normotensive pregnancies without detectable elevation in hypoxia of placental, fetal, or maternal tissue. They also show no elevation in fetal loss and their offspring are not growth impaired. Although these interspecies outcome differences strongly implicate lymphocyte-based immunity in progression of human gestational complications accompanying incomplete SA remodeling, they do not explain the physiologic mechanisms that provide normoxic placental, fetal, and maternal tissues in the absence of maternal hypertension. We now report specific cardiovascular differences between pregnant Rag2 −/− /Il2rg −/− and WT females. These differences include greater uterine arterial blood velocity and increased heart weight and performance, especially over the second half of pregnancy. These maternal adaptations are accompanied by lower fetal heart rates and reduced placental vascular space.
Materials and Methods
Mice
This study used 8-10 week old mice (39 BALB/c +/+ ; Jackson Laboratory, Bar Harbor, ME and 44 in-house bred BALB/c -Rag2 −/− /Il2rg −/− ). Timed matings were prepared using syngeneic males; copulation plug detection was dated gestational day 0. An ultrasound examination was performed once per female either before mating (nonpregnant, NP), at gestational day 2, 4, 7, 9, 10, 12, 14, 16, 18, or at 48-hour postpartum (PP). Females were then euthanized, weighed, and their organs were immediately dissected, weighed, and processed for other purposes. Wet weights were used to calculate organ to body weight (BW) ratios. Pregnancies were confirmed at gestational day 2-4 by postmortem embryo flushing and at gestational day 7-18, by viable implantation site visualization. Mean litter sizes in this study were not significantly different between genotypes. All procedures were conducted under Animal Utilization Protocols approved by the Animal Care Committee, Queen’s University.
Ultrasound procedures
Anesthetized (inhaled isoflurane) mice, after fur removal (Nair; Church & Dwight Co., Inc, Princeton, NJ), were taped (Transpore; 3M, Maplewood, MN) onto the instrument platform. A thick layer of prewarmed coupling gel (Ecogel 100; ECO-MED Pharmaceutical, Mississauga, Ontario, Canada) was applied over the area to be imaged using a 40-MHz transducer probe (Vevo 770; VisualSonics Inc, Toronto, Ontario, Canada). Body temperature was maintained at 36-37°C by the warmed platform. Maternal heart and respiration rates (ECG/Tm/Resp) were collected with a physiological controller unit.
For maternal uterine artery (UtA) power Doppler scans, bladder was first identified, then the probe was moved to locate the uterine artery arising from the common iliac. SA flows, characterized by pulsed waveforms, were imaged at the mesometrial decidual edge in 2-4 implantation sites of each gestational day 10-18 pregnancy. Peak systolic velocities of fetal umbilical arteries (UmA) were recorded before these arteries entered the placenta and branched into chorionic plate arteries (CPA) that run across the placental surface and branch into the major intraplacental arteries (IPA) in developing primary villi. After UmA study, peak systolic velocity of CPA and IPA flows were recorded in 3-5 locations/implantation sites. Maternal left renal artery (RA) flow velocity waveform was recorded to assess systemic blood velocity. For maternal data, 3-6 different females of each genotype were imaged per time-point (ie, no repeated study was made on any animal). For conceptus measurements, 6-15 implantation sites were averaged per presented time-point. Waveforms were analyzed offline.
Motion modulation (M-mode) images were constructed to evaluate maternal heart chamber dimensions at various times throughout the cardiac cycle. Three-dimensional (3-D) power Doppler data were used to estimate placental volume and structure. The Doppler beam angle was set at 27° to acquire consistent data. Doppler velocity waveforms from UtA, SA, UmA, RA, IPA, and CPA were captured using brightness mode (B-mode) imaging with the following settings: pulse repetition frequency, 10 kHz; wall filter, 100 Hz; display window, 2000 ms; sound speed, 1540 m/s; Doppler gain, 5.00 dB. The display range of the 3-D Doppler measurements of placental volume and vascular density was 19 dB (minimum)-30 dB (maximum).
Data management and analysis
Instrument software packages provided the analyses. Peak systolic velocity, mean velocity, and end diastolic velocity were measured from Doppler images. Doppler indices calculated to interpret the data were:
pulsatility index (PI) = (peak systolic velocity – end diastolic velocity)/mean velocity;
resistance index (RI) = (peak systolic velocity – end diastolic velocity)/(peak systolic velocity);
S/D ratio = peak systolic velocity/end diastolic velocity.
Cardiac measurements were obtained from M-mode data ( Supplementary Figure 4 ). Data are presented as mean (± standard deviation [SD]). Comparisons were performed by Student t test or 1-way analysis of variance (ANOVA) where appropriate for SPSS 13 analysis (SPSS Inc, Chicago, IL). A P value of less than .05 was considered significant.
Results
Maternal circulations in WT and Rag2 −/− /Il2rg −/− pregnancies
1. RA flow patterns of WT and Rag2 −/− /Il2rg −/− mice before, during, and after pregnancy . To evaluate the systemic circulation, maternal RA velocity was studied in WT and Rag2 −/− /Il2rg −/− mice (summarized data, Figure 1 , A ; full data, Supplementary Figure 1 ). RA flow velocity was similar in both strains before mating, suggesting similar homeostatic states. For both strains, RA blood velocity increased at gestational day 2 ( P < .05), remained statistically stable to gestational day 7 and dropped to a nadir at gestational day 10, the first day of placental circulation. Velocities then rebounded with a slightly delayed time course in Rag2 −/− /Il2rg −/− (peak at gestational day 12 in WT; gestational day 14 in Rag2 −/− /Il2rg −/− [ P < .05, compared with other gestational days]). RA PIs rose after conceptus implantation and peaked at gestational day 9-12 in WT ( P < .05, compared with NP). In Rag2 −/− /Il2rg −/− , the peak RA PI was delayed to gestational day 14-18 ( Figure 1 , B). At matched time points, there was no statistical difference in RA PI between the 2 strains ( Figure 1 , B).
