Video_1_Prediction of Ovarian Follicular Dominance by MRI Phenotyping of Hormonally Induced Vascular Remodeling.AVI

In the mammalian female, only a small subset of ovarian follicles, known as the dominant follicles (DFs), are selected for ovulation in each reproductive cycle, while the majority of the follicles and their resident oocytes are destined for elimination. This study aimed at characterizing early changes in blood vessel properties upon the establishment of dominance in the mouse ovary and application of this vascular phenotype for prediction of the follicles destined to ovulate. Sexually immature mice, hormonally treated for induction of ovulation, were imaged at three different stages by dynamic contrast-enhanced (DCE) MRI: prior to hormonal administration, at the time of DF selection, and upon formation of the corpus luteum (CL). Macromolecular biotin-bovine serum albumin conjugated with gadolinium-diethylenetriaminepentaacetic acid (b-BSA-GdDTPA) was intravenously injected, and the dynamics of its extravasation from permeable vessels as well as its accumulation in the antral cavity of the ovarian follicles was followed by consecutive T1-weighted MRI. Permeability surface area product (permeability) and fractional blood volume (blood volume) were calculated from b-BSA-GdDTPA accumulation. We found that the neo-vasculature during the time of DF selection was characterized by low blood volume and low permeability values as compared to unstimulated animals. Interestingly, while the vasculature of the CL showed higher blood volume compared to the DF, it exhibited a similar permeability. Taking advantage of immobilized ovarian imaging, we combined DCE-MRI and intravital light microscopy, to reveal the vascular properties of follicles destined for dominance from the non-ovulating subordinate follicles (SFs). Immediately after their selection, permeability of the vasculature of DF was attenuated compared to SF while the blood volume remained similar. Furthermore, DFs were characterized by delayed contrast enhancement in the avascular follicular antrum, reflecting interstitial convection, whereas SFs were not. In this study, we showed that although DF selection is accompanied by blood vessel growth, the new vasculature remained relatively impermeable compared to the vasculature in control animal and compared to SF. Additionally, DFs show late signal enhancement in their antrum. These two properties may aid in clinical prediction of follicular dominance at an early stage of development and help in their diagnosis for possible treatment of infertility.</p

Data_Sheet_1_Prediction of Ovarian Follicular Dominance by MRI Phenotyping of Hormonally Induced Vascular Remodeling.pdf

In the mammalian female, only a small subset of ovarian follicles, known as the dominant follicles (DFs), are selected for ovulation in each reproductive cycle, while the majority of the follicles and their resident oocytes are destined for elimination. This study aimed at characterizing early changes in blood vessel properties upon the establishment of dominance in the mouse ovary and application of this vascular phenotype for prediction of the follicles destined to ovulate. Sexually immature mice, hormonally treated for induction of ovulation, were imaged at three different stages by dynamic contrast-enhanced (DCE) MRI: prior to hormonal administration, at the time of DF selection, and upon formation of the corpus luteum (CL). Macromolecular biotin-bovine serum albumin conjugated with gadolinium-diethylenetriaminepentaacetic acid (b-BSA-GdDTPA) was intravenously injected, and the dynamics of its extravasation from permeable vessels as well as its accumulation in the antral cavity of the ovarian follicles was followed by consecutive T1-weighted MRI. Permeability surface area product (permeability) and fractional blood volume (blood volume) were calculated from b-BSA-GdDTPA accumulation. We found that the neo-vasculature during the time of DF selection was characterized by low blood volume and low permeability values as compared to unstimulated animals. Interestingly, while the vasculature of the CL showed higher blood volume compared to the DF, it exhibited a similar permeability. Taking advantage of immobilized ovarian imaging, we combined DCE-MRI and intravital light microscopy, to reveal the vascular properties of follicles destined for dominance from the non-ovulating subordinate follicles (SFs). Immediately after their selection, permeability of the vasculature of DF was attenuated compared to SF while the blood volume remained similar. Furthermore, DFs were characterized by delayed contrast enhancement in the avascular follicular antrum, reflecting interstitial convection, whereas SFs were not. In this study, we showed that although DF selection is accompanied by blood vessel growth, the new vasculature remained relatively impermeable compared to the vasculature in control animal and compared to SF. Additionally, DFs show late signal enhancement in their antrum. These two properties may aid in clinical prediction of follicular dominance at an early stage of development and help in their diagnosis for possible treatment of infertility.</p

Video_2_Prediction of Ovarian Follicular Dominance by MRI Phenotyping of Hormonally Induced Vascular Remodeling.AVI

In the mammalian female, only a small subset of ovarian follicles, known as the dominant follicles (DFs), are selected for ovulation in each reproductive cycle, while the majority of the follicles and their resident oocytes are destined for elimination. This study aimed at characterizing early changes in blood vessel properties upon the establishment of dominance in the mouse ovary and application of this vascular phenotype for prediction of the follicles destined to ovulate. Sexually immature mice, hormonally treated for induction of ovulation, were imaged at three different stages by dynamic contrast-enhanced (DCE) MRI: prior to hormonal administration, at the time of DF selection, and upon formation of the corpus luteum (CL). Macromolecular biotin-bovine serum albumin conjugated with gadolinium-diethylenetriaminepentaacetic acid (b-BSA-GdDTPA) was intravenously injected, and the dynamics of its extravasation from permeable vessels as well as its accumulation in the antral cavity of the ovarian follicles was followed by consecutive T1-weighted MRI. Permeability surface area product (permeability) and fractional blood volume (blood volume) were calculated from b-BSA-GdDTPA accumulation. We found that the neo-vasculature during the time of DF selection was characterized by low blood volume and low permeability values as compared to unstimulated animals. Interestingly, while the vasculature of the CL showed higher blood volume compared to the DF, it exhibited a similar permeability. Taking advantage of immobilized ovarian imaging, we combined DCE-MRI and intravital light microscopy, to reveal the vascular properties of follicles destined for dominance from the non-ovulating subordinate follicles (SFs). Immediately after their selection, permeability of the vasculature of DF was attenuated compared to SF while the blood volume remained similar. Furthermore, DFs were characterized by delayed contrast enhancement in the avascular follicular antrum, reflecting interstitial convection, whereas SFs were not. In this study, we showed that although DF selection is accompanied by blood vessel growth, the new vasculature remained relatively impermeable compared to the vasculature in control animal and compared to SF. Additionally, DFs show late signal enhancement in their antrum. These two properties may aid in clinical prediction of follicular dominance at an early stage of development and help in their diagnosis for possible treatment of infertility.</p

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

Arterial delivery of maternal blood to the placentas is dependent on litter size and on the position of the placenta along the uterine horn.

<p>(A) Mean fluorescence signal in placentas of a uterine horn after ligation of the left uterine a. (artery), right uterine a., left ovarian a., or right ovarian a. (n = 5 animal/group). For each animal, values were normalized (%) to the mean fluorescence signal of the placentas in the contralateral, non-ligated horn (mean ± SEM; a,b significant differences: <i>P</i><0.05). (B) Mean fluorescence signal in placentas of a uterine horn after unilateral ligation of the uterine or ovarian arteries (n = 10 animal/group, left and right sides combined). For each animal, values were normalized (%) to the mean fluorescence signal of the placentas in the contralateral non-ligated horn (mean ± SEM; a,b significant differences: <i>P</i><0.05). (C) Overall relative contributions of the uterine and ovarian arteries to the mean fluorescence signal in placentas in a uterine horn. Data are presented as Box-and-whisker plots, with the median, the 25% and 75% percentile ranges (box depth), and the maximum and minimum (T-bars). Significant differences (a, b; <i>P</i><0.05). (D) Relative contributions of the uterine and ovarian arteries to the fluorescence signal in placentas located in different positions along the horn. Data were extracted only from symmetric pregnancies that contained 4 fetuses in each horn (n = 5 dams). (E) Relative contributions of the uterine and ovarian arteries to the fluorescence signal in placentas located in different positions along the horn. Data were extracted only from symmetric pregnancies that contained 6 fetuses in each horn (n = 4 dams).</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

Numerical simulation of placental function in the pregnant mouse reveals a possible hemodynamic basis for the observed ‘position dependence’ and ‘neighbor effects’.

<p>(A) Simulation performed for the case of five fetuses in a single uterine horn and a hypothetical case, in which the resistance along the uterine and ovarian arteries was very low. Note the flattening of the U-shaped pattern in the latter case. (B) Simulation of the effect of increasing litter size with a fixed battery voltage V_H (no cardiac output compensation)_. (C) Simulating a case of nine ‘fetuses/placentas’ with adjustment of V_H (i.e., adjustment of cardiac output) to maintain the same average I_p as in the case of five fetuses per uterine horn. (D) Ligation simulations of the uterine artery showing I_p of placentas in the non-ligated and ligated horns, normalized to the average I_p of the non-ligated horn. (E) Ligation simulations of the uterine branch of the ovarian artery showing I_p of placentas in the non-ligated and ligated horns, normalized to the average I_p of the non-ligated horn. (F) Simulation of the placenta of a fetus that spontaneously died during mid pregnancy. The current flowing in normal placentas located closest to the dead fetus is compared to placentas located distant from the dead fetus. The dead fetus was simulated by reduced placental resistance compared to normal placentas. (G) Simulation of <i>Akt1</i>^−/− placenta; the average I_{p,<i>Akt1+/+</i>} of <i>Akt1</i>^+/+ having a <i>Akt1</i>^−/− neighbor compared with <i>Akt1</i>^+/+ placentas having only <i>Akt1</i>^+/+ neighbors. The <i>Akt1</i>^−/− placenta was simulated as increased resistance compared to <i>Akt1</i>^+/+ placentas.</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

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