The Hemodynamic Basis for Positional- and Inter-Fetal Dependent Effects in Dual Arterial Supply of Mouse Pregnancies

<div><p>In mammalian pregnancy, maternal cardiovascular adaptations must match the requirements of the growing fetus(es), and respond to physiologic and pathologic conditions. Such adaptations are particularly demanding for mammals bearing large-litter pregnancies, with their inherent conflict between the interests of each individual fetus and the welfare of the entire progeny. The mouse is the most common animal model used to study development and genetics, as well as pregnancy-related diseases. Previous studies suggested that in mice, maternal blood flow to the placentas occurs via a single arterial uterine loop generated by arterial-arterial anastomosis of the uterine artery to the uterine branch of the ovarian artery, resulting in counter bi-directional blood flow. However, we provide here experimental evidence that each placenta is actually supplied by two distinct arterial inputs stemming from the uterine artery and from the uterine branch of the ovarian artery, with position-dependent contribution of flow from each source. Moreover, we report significant positional- and inter-fetal dependent alteration of placental perfusion, which were detected by in vivo MRI and fluorescence imaging. Maternal blood flow to the placentas was dependent on litter size and was attenuated for placentas located centrally along the uterine horn. Distinctive apposing, inter-fetal hemodynamic effects of either reduced or elevated maternal blood flow, were measured for placenta of normal fetuses that are positioned adjacent to either pathological, or to hypovascular <em>Akt1</em>-deficient placentas, respectively. The results reported here underscore the critical importance of confounding local and systemic in utero effects on phenotype presentation, in general and in the setting of genetically modified mice. The unique robustness and plasticity of the uterine vasculature architecture, as reported in this study, can explain the ability to accommodate varying litter sizes, sustain large-litter pregnancies and overcome pathologic challenges. Remarkably, the dual arterial supply is evolutionary conserved in mammals bearing a single offspring, including primates.</p> </div

Assessment of the arterial blood supply to the placenta via BD-ASL MRI and intravital fluorescence microscopy.

<p>Two methods were used to explore the pattern of transfer of arterial blood to the placentas along the uterine horns in pregnant mice at late gestation (E17.5): 1) Bi-directional ASL methodology (Panels A–C); and 2) Intravital fluorescence microscopy imaging of the uterine arterial blood supply subsequent to intravenous administration of FITC-dextran to mice having undergone surgical arterial ligations of either the uterine branch of the ovarian artery, or the uterine artery (Panels D–F). (A–C) Placental saturation transfer maps obtained by BD-ASL MRI of an ICR pregnant mouse (E17.5). For placentas positioned closer to the cervix (Panel A: L1, R1), mainly negative BD-ASL contrast voxels (blue) were observed, consistent with the predominant contribution of maternal blood flow through the uterine artery. In placentas closer to the ovary (Panel C: L5–7), the BD-ASL contrast was mainly of positive voxels (red), implying that placentas in this part of the uterine horn are supplied through blood mainly from the uterine branch of the ovarian artery. Placentas located in the central region of the uterine horn (Panel B, L3–4) had a dispersive pattern of BD-ASL values with both negative and positive voxels, consistent with a dual supply from both the uterine artery and the uterine branch of the ovarian artery, respectively. (D) Intravital fluorescence microscopy image of the arterial blood supply to an intact uterine horn (a snapshot from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052273#pone.0052273.s003" target="_blank">Movie S1</a>). (E) Intravital fluorescence microscopy image of the arterial blood supply to a uterine horn following ligation of the uterine artery (a snapshot from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052273#pone.0052273.s004" target="_blank">Movie S2</a>). (F) Intravital fluorescence microscopy image of the arterial blood supply to a uterine horn following ligation of the uterine branch of the ovarian artery (a snapshot from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052273#pone.0052273.s005" target="_blank">Movie S3</a>).</p

Placenta of <i>Akt1^+/+</i> positioned adjacent to an <i>Akt1^−/−</i> placenta/fetus showed increased |BD-ASL| and PBVm.

<p>(A) |BD-ASL| values in placentas of fetuses of different <i>Akt1</i> genotypes (mean ± SEM; a, b, <i>P</i><0.05). (B) Fluorescence values and PBVm values in placentas of fetuses of different <i>Akt1</i> genotypes (mean ± SEM; a, b, <i>P</i><0.05). (C) |BD-ASL| values in <i>Akt1^+/+</i> placentas located near an <i>Akt1^−/−</i> fetus, as compared to <i>Akt1</i>^+/+ placentas not located near an <i>Akt1^−/−</i> fetus (mean ± SEM; <i>P</i> = 0.0712). (D) Fluorescence values and PBVm values in <i>Akt1^+/+</i> placentas located near a <i>Akt1^−/−</i> fetus, as compared to <i>Akt1^+/+</i> placentas not located near an <i>Akt1^−/−</i> fetus (mean ± SEM; a, b, <i>P</i><0.05).</p

Electrical circuit modeling of the hemodynamics of maternal arterial supply in the mouse pregnancy.

<p>(A) Diagram of mouse uterine horns and their arterial blood vessels in the gestation period after placentas have formed (E10.5 to term). Our results demonstrate that each placenta can be perfused from two maternal arteries. F Fetus, K Kidney, Ov Ovary, Pl Placenta, Ut Uterine. (B) Schematic diagram of mouse placenta. Dec Decidua, Sp Spongiothrophoblast, TGC Throphoblast Giant Cells, Lab Labyrinth. (C) Numerical simulation of blood flow in multi-fetus pregnancy modeled as an electrical circuit. Bi-directional blood flow in each of two uterine horns was modeled by the respective currents: I_ua(l/r) for the left or right uterine arteries and I_oa(l/r) for the left or right uterine branch of the ovarian artery. The balance between I_ua(l/r) and I_oa(l/r) was set at 3∶1 using resistors placed into the circuit near the battery. Resistance to flow along the uterine branch of the ovarian artery and the uterine artery was modeled by a series of identical resistors (one per implantation site). (D) Placenta modeled as an electrical circuit. Resistance to flow into the placenta via the spiral arteries was modeled by low-value resistors, which were connected separately to a second resistor to ground, simulating exchange within the placenta itself and the flow back to ground (i.e., clearance of blood through the maternal veins). Diffusion across the placenta was modeled by the insertion of a large resistor (20× the value of the dual resistors to ground representing flow into the placenta), between the dual arterial input resistors. Total maternal blood supply to each placenta I_p,i was therefore derived from I_p,oa,i+I_p,ua,i. Arrows indicate the direction of flow.</p

Position dependence of maternal blood to placentas along the uterine horn.

<p>(A) Experimental scheme for multi-modal functional imaging of pregnant mice: pregnant female ICR mice (E17.5) were analyzed using MRI, intravital fluorescence microscopy, and ex vivo fluorescence analysis of the maternal blood volume in the placenta (PBVm). Note fetuses (F) and their placentas (white arrow heads). (B) Data for a pregnant mouse carrying 5 fetuses in one uterine horn. Position dependence of maternal bi-directional perfusion was detected by MRI (|BD-ASL|). (C) Ex vivo fluorescence and corresponding PBVm values for the placentas in one uterine horn, in the same pregnant mouse as in A. (D) Correlation between PBVm and |BD-ASL| (n = 11 dams, 86 placentas/fetuses; r = 0.62, <i>P</i><0.0001). (E) Correlation between PBVm and fetal body weight (n = 11 dams, 86 placentas/fetuses; r = 0.64, <i>P</i><0.0001).</p

Intra-uterine neighbor effect: reduced |BD-ASL| and PBVm in placenta positioned adjacent to a pathological/dead fetus.

<p>(A) A representative example from one animal; placental |BD-ASL| (upper panel), and placental fluorescence signal with the corresponding PBVm values (lower panel) of placentas along a uterine horn that contains one pathological fetus (#1, marked in red). The upper image presents the pathological fetus and its placenta adjacent to a normal fetus and placenta (fetus #3). Note the reductions in |BD-ASL| and PBVm values in the placenta of the pathological fetus, as well as in the adjacent placenta (#2). (B) Mean |BD-ASL| values in placentas of pathological/dead fetuses, fetuses positioned adjacent to pathological/dead fetus, and normal fetuses located far from a pathological/dead fetus. Note that mean |BD-ASL| value is significantly lower for those fetuses that have a pathological/dead neighbor. Different letters above bars indicate significant differences (mean ± SEM; a,b, <i>P</i><0.05) (C) Fluorescence values and corresponding PBVm values in placentas of pathological/dead fetuses, fetuses located near a pathological/dead fetus, and normal fetuses located distant from a pathological/dead fetus. Note that mean fluorescence value and PBVm values are significantly lower for those fetuses that have a pathological/dead neighbor (mean ± SEM; a,b, <i>P</i><0.05).</p

Contribution of arterial blood supply to the placenta.

<p>Arterial ligations were performed on ICR pregnant mice (E17.5) for either the uterine branch of the ovarian artery (n = 10 mice, 5 mice on each side; 82 placentas/fetuses); or in the uterine artery (n = 10 mice, 5 mice on each side; 92 placentas/fetuses). (A, B) Relative fluorescence signal in placentas along two uterine horns upon ligation of the left (A) or the right (B) uterine artery (each graph presents data from one animal. orange: ligated uterine horn; green: non-ligated uterine horn). (C, D) Relative fluorescence signal in placentas along two uterine horns upon ligation of the left (C) or right (D) ovarian artery (each graph presents data from one animal). (E, F) Histological sections of the placentas along two uterine horns upon ligation of the right uterine artery (E; same animal as in panel B) or right ovarian artery (F; same animal as in panel (D) Left) H&E section. Right) fluorescence microscopy (right; Green = FITC-dextran; Blue =  DAPI; inset fluorescent image of the ex vivo placenta).</p

MOESM3 of Microbial communities and inflammatory response in the endometrium differ between normal and metritic dairy cows at 5–10 days post-partum

Additional file 3. Rarefaction curves of 16S-rDNA pyrosequencing endometrial samples from healthy, metritic and septic metritic cows. Rarefaction curves of the number of OTUs are presented as a function of read number. H - healthy cows, endometrial swab samples; M - metritic cows, endometrial swab samples; BM - metritic cows, full-thickness uterine biopsy samples; BS - septic metritis cow, full-thickness uterine biopsy sample

MOESM5 of Microbial communities and inflammatory response in the endometrium differ between normal and metritic dairy cows at 5–10 days post-partum

Additional file 5. Most abundant genera of the phyla Bacteroidetes, Firmicutes and Fusobacteria in healthy and metritic cows with similar community composition. Samples were collected by endometrial swabs at 5–10 days post-partum and used for 16S-rDNA pyrosequencing analysis. For each phylum, the ten most abundant OTUs were included in descending order. Some genera appear twice or more as distinct OTUs. *The healthy group is composed of four cows with a similar bacterial community composition to the average metritic cow community composition sorted by phylum

MOESM4 of Microbial communities and inflammatory response in the endometrium differ between normal and metritic dairy cows at 5–10 days post-partum

Additional file 4. Endometrial bacterial community composition by phylum in healthy and metritic cows. Samples were collected by endometrial swabs at 5–10 days post-partum and used for 16S-rDNA pyrosequencing analysis. Results are presented as mean abundance ± SEM. P value is specified where a statistically significant difference was found between the groups; NS: not significant