Method of assessing placentation

An improved method of visualizing blood flow in the maternal placenta is provided. By utilizing contrast media to enhance the ultrasound image, the condition of the maternal placenta during pregnancy can be evaluated so as to permit intervention for correcting abnormal conditions of such blood flow, if required.

FIELD OF THE INVENTION 
The present invention relates to the assessment of placental function in as 
early as the second trimester of pregnancy. In particular, the invention 
relates to the use of contrast media to enhance the ultrasound detection 
of blood flow in a placenta during pregnancy. 
BACKGROUND ART 
The placenta is a remarkable fetal support system. It functions as the 
fetal lung and is responsible for all gas transfer from the mother to the 
fetus. The placenta controls excretory functions, water balance, and pH 
regulation as the fetal kidney. It acts as the fetal gut by performing 
catabolic and resorptive functions. It also performs most synthetic and 
secretory functions of the endocrine glands and liver. Finally, the 
placenta provides hematopoiesis of the bone marrow during the early stages 
of pregnancy, controls heat transfer of the skin, and performs 
immunological functions to a largely unknown degree. 
Disruptions in the maternal circulation to the placenta, and thus in the 
fetal circulation, can result in intrauterine growth retardation ("IUGR") 
or even fetal death. IUGR may be a direct effect of hypoxia upon fetal 
tissues or caused by a reduction in the supply of nutrients to the fetus. 
The importance of uteroplacental blood flow in the nutrition of both the 
maternal and fetal placenta has been shown by observations of placentas in 
animal models and in fetuses that have not survived IUGR. Histological 
characteristics used by investigators to describe placental insufficiency 
have been correlated to the consequences of fetal nutritional deprivation. 
As many as 250,000 babies are born each year that weigh less than 2,500 
grams and are thus described as low birth weight (LBW) infants. These 
babies are 40 times more likely to die in the neonatal period. For the 
segment of newborns weighing less than 1,500 grams, the relative risk is 
200 times greater. In 1990, the hospital-related costs of caring for all 
LBW infants during the neonatal period totaled more than two billion 
dollars. LBW infants make up only about 7% of all births, but the costs 
associated with their care in 1990 represented about 57% of the costs 
incurred for all newborns. 
Even when LBW infants survive, about half are categorized as "small for 
their gestational age" ("SGA"). These SGA infants are significantly 
compromised in utero and lack the resilience normally seen for infants at 
their gestational age during labor, delivery and the neonatal period. 
Sometimes, infants are even classified as both premature and SGA, and 
these babies constitute a significant minority that have an even higher 
morbidity rate. Epidemiological studies have shown that it is the relative 
weight of the infant, rather than the length of gestation, that is the 
primary factor affecting an infant's chances of survival. 
Additionally, pathological alterations in the placental vasculature have 
been correlated to preeclampsia, a disease affecting 5 to 10% of pregnant 
women. Left undetected, women with preeclampsia are at risk for maternal 
death, fetal IUGR and fetal death. 
There has been a need to reduce perinatal morbidity and mortality by 
significantly reducing the incidence of both the SGA fetus and the LBW 
fetus. To this end, there is a need to protect human fetuses that are at 
risk by assuring a maternal environment that can support fetal nutritional 
needs. 
One way to do this is to attempt to visualize the maternal circulation in 
the placenta in a sonogram. However, current methods that employ 
ultrasound techniques including Doppler flow to examine intervillous blood 
flow of the mother's circulation in the placenta have not been entirely 
successful. This is because current ultrasound techniques are limited by 
the low flow state, the relatively small target spaces, and the lack of 
spatial resolution. 
Contrast media have been suggested to detect aspects of flow and tissue 
perfusion in other situations, most frequently, the heart or the organs of 
the gastrointestinal tract, but also including the cardiovascular system, 
liver, spleen, kidney, pancreas, tumor tissue, muscle tissue, or bodily 
fluids such as blood. See, for example, Unger, U.S. Pat. No. 5,209,720 
issued May 11, 1993 (tumor tissue, muscle tissue or blood fluid), and 
Unger et al., U.S. Pat. No. 5,228,446 issued Jul. 20, 1993 (liver, spleen, 
kidneys, heart, vasculature, diseased tissue and blood flow). 
More specifically, Erbel et al., U.S. Pat. No. 5,205,287 issued 27 Apr. 
1993, states that certain ultrasonic contrast media can be used to 
visualize the blood flow in the right ventricle or left side of the heart, 
the myocardium, the liver, spleen, kidney or brain. Further, the contrast 
media of Erbel et al. are described as also suitable for the visualization 
of the urinary bladder, ureter, uterus or vagina. Two U.S. patents (Rasor 
et al. U.S. Pat. No. 4,442,843 and Rasor et al., U.S. Pat. No. 4,681,119) 
describe the use of contrast media to enhance ultrasonic images of a 
number of liquid-filled regions of the body, such as those of the 
reproductive system. 
Others have disclosed the use of an intrauterine catheter containing a 
contrast medium in conjunction with a diagnostic scanning device. For 
example, Eden, U.S. Pat. No. 4,349,033 issued Sep. 14, 1982, discloses a 
method of providing a fluid to the interior of a body cavity, such as 
woman's uterus, while scanning with an ultrasonic transducer. 
Visualization of pelvic structures, the Fallopian tubes, ovaries and the 
cul-de-sac is said to be improved. The technique is described as allowing 
a doctor to detect the presence of a pelvic mass, abnormal vaginal 
bleeding, pelvic cancer staging, congenital anomalies or pelvic 
infections. 
Two investigators have described the use of an ultrasound contrast medium 
to evaluate the condition of Fallopian tubes. Unger, U.S. Pat. No. 
5,230,882 issued Jul. 27, 1993, discloses a general method for imaging a 
patent using ultrasound comprising administering to the patient a liposome 
contrast medium and scanning the patient using an ultrasound device. In a 
nonvascular application, the liposomal contrast medium may be injected 
directly into the area to be scanned, such as into the uterine cavity to 
assess "patency" of the Fallopian tubes. According to the second 
investigator, in the second document, Soviet Union Patent Document No. 
1323082, published Jul. 15, 1987, "uterine tube patency" may be diagnosed 
by filling the tubes with a spasmolytic solution, followed by ultrasound 
visualization. However, there is no disclosure of using ultrasound during 
pregnancy, let alone a teaching of applicability to the placenta. 
As to the use of an ultrasound contrast medium during pregnancy for other 
purposes, Finberg et al., in an article entitled "Definitive Prenatal 
Diagnosis of Monoamniotic Twins: Swallowed Amniotic Contrast Agent 
Detected in Both Twins on Sonographically Selected CT Images", J 
Ultrasound Med (1991) 10:513-16, describes the diagnosis of the 
potentially dangerous condition of monoamniotic twinning at 27 weeks. This 
diagnosis was by the detection, on sonographically selected computed 
tomographic ("CT") images, of intestinal opacification in each twin after 
a single injection of the contrast medium into the amniotic space. FIGURE 
1 of the publication, which appears to be a conventional sonogram without 
the use of contrast media, is described as showing the positions where the 
umbilical cords of both twins insert into the placenta and the absence of 
a detectable intervening amniotic membrane. When a single amniocentesis is 
performed to inject a contrast medium into the amniotic fluid, which is 
then swallowed by each twin, the ultrasound-selected CT images showed 
concentrated contrast medium in the intestine of each twin, confirming 
that they were monoamniotic. When this condition was confirmed, labor was 
induced to deliver the twins prematurely to prevent catastrophic events 
due to the knotting of intertwined loops of the two umbilical cords 
sharing the same amniotic sac. 
However, there is no teaching in Finberg et al. of how to evaluate the 
condition of the placenta itself, or the volume of blood flowing through 
the placenta, even in this environment, much less in instances of 
apparently normal pregnancies. 
The literature in this area can thus be divided roughly into two groups: 
(1) the use of ultrasound contrast media to assess the condition of 
various body cavities, such as parts of the reproductive system, for 
example, the uterus, Fallopian tubes and/or vagina; (2) and the use of 
ultrasound scanning with a contrast medium during pregnancy to confirm a 
suspected diagnosis of monoamniotic twins. 
Moreover, those working in the art have not proposed the use of contrast 
media to facilitate the ultrasound assessment of the condition of the 
placenta itself during pregnancy. The use of contrast media for direct 
observation of the fetus has generally been ruled out as involving an 
undue risk, particularly in monitoring what is an apparently healthy 
pregnancy. Those working in the ultrasound art have expressly avoided 
fetal-prenatal vascular studies altogether due to the prevailing fears of 
introducing complications during pregnancy. 
Therefore, there remains a need for a method to study maternal intervillous 
and related placental blood flow that allows for a measure of 
uteroplacental circulation. In this way, it is proposed that poor 
placentation can be detected at a sufficiently early stage to attempt 
treatment. 
DISCLOSURE OF THE INVENTION 
It has now been discovered that contrast media can be used to enhance the 
ultrasound detection of blood flow in a placenta during the pregnancy of 
an animal. Specifically, abnormalities in the placenta of a pregnant, 
female animal may be detected by a method for evaluating blood flow in the 
placenta comprising the steps of: 
a. administering to the animal an efficacious amount of a contrast medium; 
and 
b. scanning the abdomen of the animal with the transducer of an ultrasound 
scanner; and 
c. assessing the condition of the placenta by evaluating the scan. 
The use of ultrasound contrast media allows greater backscatter and thus 
augments the ability of current ultrasound techniques to image the blood 
flow found in the intervillous space of an animal's placenta. Further, 
when ultrasound sonograms are enhanced by the use of appropriate contrast 
media, abnormalities can be detected early enough for therapeutic 
intervention, resulting in a lower perinatal morbidity and mortality rate.

MODES OF CARRYING OUT THE INVENTION 
The method of the invention is applicable to any animal that is a 
"placental" - - - i.e., that nurtures the unborn fetus through a placenta. 
Such animals include humans, other primates, mammalian food animals and 
pets. 
One purpose of the invention is to allow the measurement of maternal blood 
flow of such animals into the intervillous space. It is the maternal blood 
in the intervillous space that constantly bathes the fetal villi, and it 
is through the fetal villi that nutrients are delivered into fetal 
circulation. 
From the mother's heart descends the aorta, and the terminal branches of 
the aorta are the common iliac arteries. Arising opposite the fourth 
lumbar vertebrae, they pass downward and laterally to end at the 
lumbosacral junction by dividing into internal and external iliac 
arteries. The internal iliac arteries descend into the pelvis and give off 
the uterine artery, which supplies the uterus and separates into arcuate 
arteries. Each arcuate artery subdivides hundreds of times into tiny 
spiral arteries. These very small arteries feed into the intervillous 
space. It is this blood flow from the mother's arcuate arteries into the 
spiral arteries and into the intervillous space that provides important 
information regarding the nutritional condition of the fetus. 
Postpartum to an IUGR pregnancy, analysis of the placenta in a pathology 
lab typically reveals signs of pathology along the spiral arteries, which 
tend to hinder or even obstruct the blood flowing from the mother's spiral 
arteries into the intervillous space. Usually, the spiral arteries are 
significantly narrowed or hardened and may even become completely clogged. 
Using conventional ultrasound techniques, the blood flow in the spiral 
arteries cannot be visualized because it is simply too small and there are 
literally hundreds of them, such that examining each one of them 
independently would be out of the question. Thus, it would not be 
possible, let alone practical, to attempt to sample each and every spiral 
artery to determine whether or not blood is flowing freely through it. 
While those in the art have long desired the ability to visualize or 
measure the blood flow into the intervillous space, the problem has been 
that blood flow, even in normal placentation, is too slow to achieve 
acceptable resolution in conventional Doppler sonography. Occasionally, 
particularly talented sonographers may have been able to get a fleeting 
visible image, but such an image has not been produced reproducibly. 
In the claimed invention, the use of an appropriate contrast medium allows 
visualization of the rate and direction of blood flow in the arcuate 
arteries, spiral arteries and intervillous space by Doppler ultrasound 
examination. A contrast medium is administered appropriately, such as, for 
example, by injection into one of the mother's veins or arteries. The 
Doppler transducer is fixed on the mother's abdomen and focused on the 
desired location related to the placenta, such as intervillous space. The 
gain of the transducer is then changed to produce a low intensity signal, 
so that very little motion or activity is seen on the visual display, such 
as a CRT or liquid crystal screen. 
When the contrast medium is introduced, the flushing of the medium through 
the placenta can be observed as a lighting up of the intervillous space, 
somewhat like a flare or firecracker. This flare phenomenon is called a 
"bloom." Then the bloom caused by the medium fades away as the medium is 
washed out of the region. 
An important element in an ultrasound system is the scanner transducer. The 
transducer converts electrical to mechanical energy and vice versa. An 
ultrasound scanner, under computer control, sends a short pulse of 
electricity to the transducer to generate a burst of mechanical vibrations 
(sound waves). The sound generated has a frequency above the audible limit 
of 20 kHz and, in clinical applications, is usually in the range of 2 to 
10 MHz. The frequency of the sound waves is adjusted to accommodate the 
contrast medium used, as is generally understood in the art. 
The burst of sound travels down the tissue and reflects back. When the 
reflected wave strikes the now silent transducer, the resulting mechanical 
energy is converted back to electrical energy, which is then returned to 
the computer. The time delay between the moment the sound was generated 
and the moment a signal was received allows the computer to place a dot on 
the visual display. The brightness of the dot is related to the amplitude 
of the received signal. The computer keeps track of the position of the 
transducer (or sound beam) and its angle. The dot is positioned along the 
long axis of the transducer at a point dictated by the time delay. 
Reflection of sound in biological tissues occurs when the sound beam 
strikes an interface separating two regions with different acoustic 
impedance (the product of mass density and acoustic velocity). Since the 
difference in density between most tissues is minimal, the major 
contributor to the differences is velocity. The difference in velocity 
between most animal tissues is on the order of about 1 to 5%. 
Surfaces of organs typically reflect sound in a manner analogous to the 
reflection of light off the surface of a mirror. However, when the sound 
beam strikes a series of smaller, more diffuse reflectors, it is scattered 
in nearly all directions in a manner analogous to light reflected from a 
series of small spheres. Particles of ultrasound contrast media are 
considered diffuse reflectors or scatterers. 
Overall tissue texture is dependent upon the number of scatterers present 
in the field and the spatial relationship among them. The image is the 
final result of interactions between the sound wave and the scatterers 
that interfere with each other in a constructive or a destructive manner. 
The Doppler ultrasound effect is produced when the reflective surface is in 
motion relative to the transducer, for example, a flow of blood through a 
blood vessel or highly vascular tissues. If the reflective tissue is 
approaching the transducer, the sound waves are intercepted at a faster 
rate than if the reflective tissue were stationary, increasing the 
frequency of the reflected sound. Conversely, if the reflective tissue is 
moving away from the transducer, it would intercept the waves at slower 
rate, and the reflected sound will have a lower frequency. 
By evaluating the difference between the frequency delivered by the 
transducer and the frequency received, the velocity of the reflective 
tissue in the direction parallel to the long axis of the transducer can be 
calculated. This velocity allows one to assess the degree of flow, such as 
blood flow. A typical display permits the quantitative analysis of 
velocities observed in a single anatomic location over time. 
Color Doppler, on the other hand, is qualitative. It provides the location 
of detectable motion within the entire region of interest at one point. 
Red and blue are typically assigned to locations with frequency shifts 
that are either positive (motion towards the transducer) or negative 
(motion away from the transducer). While color Doppler is qualitative, it 
serves as a rapid survey of flow in the entire field. When quantitative 
measurements are required, color Doppler is often used first to guide the 
operator to the area of interest. 
The ultrasound contrast medium used in the method of the invention may 
comprise any one of a large number of compounds or a mixture of these 
compounds, but must eventually decompose into innocuous materials when 
administered into an animal body. 
The material properties of contrast media affect the resulting 
effectiveness of the evaluation. Typical important parameters include 
particle size, imaging frequency density, compressibility and particle 
behavior (surface tension, internal pressure, bubble-like qualities). 
Equally important are biodistribution characteristics and tolerance. 
Further, the transducer frequency, must be tuned to the medium used. 
Typical ultrasound contrast media include bubbles in a physiologically 
acceptable liquid, protein-encapsulated bubbles, solid particles 
encapsulating a gas, polymers encapsulating a gas, lipids and liposomes 
encapsulating a gas, and fluorocarbon compositions. It is well known that 
gas is highly reflective within the vascular space and tissues. Free gas 
bubbles in a physiologically acceptable liquid, which range in size from 
about 2 .mu.m to 12 .mu.m and persist from 2 or 3 to 30 seconds, have been 
used, for example, to opacify cardiac chambers. While gas-based contrast 
media are extremely reflective and produce efficacious results at small 
dosages, they tend to be short-lived in animal plasma. Echogenicity may be 
as short as several seconds and tends to provide only first pass data. 
None usually pass the pulmonary capillaries to opacify, for example, the 
left ventricle of the heart. 
Preferably, the contrast medium of the invention is persistent for more 
than a few circulation times and capable of reaching the placenta from the 
vascular tree into which it is injected. To increase the stability or 
longevity of the bubbles, gas can be encapsulated within a protective 
shell, usually a protein such as gelatin. Among the several types of 
encapsulated media, the human albumin microsphere, given the trade name 
Albunex.RTM. (Molecular Biosystems, San Diego, Calif.) is often used for 
echocardiography. Also useful are galactose particles (8HU-454.RTM. and 
SHU-508.RTM., Echovist.RTM., Schering AG, Berlin, Germany) with the gas 
being carried on the surface of the particle as an inside-out bubble, 
lipids (monolayer) encapsulating a gas, and liposomes (gas-filled lipid 
bilayer bubbles or microbubbles having a hydrophilic layer and an opposing 
hydrophobic layer). 
Thus, by changing the chemical nature of the contrast medium, the 
ultrasound bloom duration can be increased to a longer periods of time. 
Certain contrast media persist longer, causing the bloom to be visible for 
a considerable length of time. Other contrast media, being quickly 
destroyed by the animal body, cause the bloom to fade more rapidly. 
Therefore, a balance must be struck so that the half life of the contrast 
medium is sufficient to permit satisfactory observation but not so long as 
to persist in the circulation of the subject. This is one of the important 
factors affecting the choice of an appropriate contrast medium for claimed 
invention. Preferably, the contrast medium is chosen to ensure a bloom 
having a predetermined, predictable lifetime, with a lifetime between 
about five seconds and about five minutes being particularly preferred. 
Solid particles, e.g., of iodipamide ethyl ester, have been used to opacify 
internal organs. Solid particle formulations can exhibit reflectivity 
increased by as much as a factor of twenty by entrapping gas in the solid 
matrix. These formulations of solid particles encapsulating a gas show 
vascular and tissue enhancement from intravenous injections that may 
persist for as long as several minutes. 
Successful and persistent tissue opacification from intravenous infusion 
has also been achieved by the use of fluorocarbon compositions, usually 
emulsions. Visualization of flow, tissue perfusion, and Doppler 
enhancement has also been achieved with fluorocarbon emulsions 
administered intravenously for example, with Fluosol.RTM., a 20% w/v 
emulsion of perfluorooctylbromide. While fluorocarbon emulsions are 
efficacious and produce a prolonged effect (sometimes for an hour or more) 
on vascular and tissue enhancement, they are less reflective than gases 
such as air and therefore require larger dosages to produce a comparable 
echogenic effect; also they may be too persistent. Encapsulated gas 
bubbles are most preferred, usually providing both stability and good 
echogenicity. 
The reflectivity of particulate contrast media is also related to the 
particle's scattering cross-section, which in turn is related to particle 
size, the difference in the acoustic impedance of the medium and the 
surrounding environment, particle elasticity, and the frequency of the 
interrogating sound wave from the transducer. 
Of the above-listed factors, particle size has the most profound effect on 
reflectivity. Specifically, the amount of backscatter is related to 
r.sup.6, where r is the radius of the particle. However, particle size 
must be very small to successfully traverse the capillary bed. Preferably, 
the particle size of the contrast medium is less than or equal to about 8 
.mu.m, more preferably less than 7 .mu.m, such that it is small enough to 
pass through the capillary bed. 
Both the particle size and the bulk properties of the medium (acoustic 
impedance) greatly affect the scattering cross-section of the particle. 
For instance, perfluorochemicals, solid particles, and air bubbles are 7, 
10, or even 10.sup.10 times more reflective than red blood cells 
respectively. 
Further, the elasticity of the particle, which is related to the 
compressibility of the medium and the stiffness of the outer shell of the 
particle, can dramatically affect reflectivity, allowing a particle with a 
small diameter (less than 5 .mu.m) to increase in scattering cross-section 
a few orders of magnitude to become effective at commonly used clinical 
frequencies with a wavelength on the order of 200-500 .mu.m. Gas, and to a 
much lesser degree fluorocarbons, are compressible and can potentially 
exhibit a resonance phenomenon. 
Other specific characteristics that may be important in the selection of 
one or more appropriate contrast media include the distribution of bubbles 
or particles in comparison to the distribution of echoes, signal-to-noise 
ratio, and special characteristics of the echoes of the uterus, placenta, 
umbilical cord and fetal tissue. 
The contrast media of the invention may include ultrastable microbubbles, 
which can now be manufactured in a tight range of diameters (such as 1-5 
.mu.m) with a mean diameter of about 2.mu.m. These media can enhance the 
echogenicity of these tumors for at least up to 5 minutes. This medium is 
described by Simon in an article entitled "Quantitative Assessment of 
Tumor Enhancement by Ultrastable Lipid-Coated Microbubbles as a 
Sonographic Contrast Agent" in Inves Radiol (1992) 27:29-34 (1992). 
Thus, the chemical nature of the contrast medium should be such that there 
is minimal impact on the mother and the fetus, but provision of the 
desired degree of bloom and duration of bloom upon visual inspection of 
the ultrasound imaging output. 
A wide variety of contrast media is available commercially. These media 
include fluorocarbon-based formulations Imagent US.TM. (Alliance 
Pharmaceutical) and Echogen.TM. (Sonus Pharmaceutical); cellulose or other 
carbohydrate formulations such as SonoR.sub.x .TM. (ImaR.sub.x 
Pharmaceutical) MB-U820.TM. (Molecular Biosystems), Echovist.TM. and 
Levovist.TM. (Schwing AG), liposomal compositions such as Aerosomes.TM. 
(ImaR.sub.x .TM.) and Filmix.TM. (Cavcon); protein microspheres such as 
Albunex.TM. (Molecular Biosystems) and iodinated polymeric microparticles 
called "bubbles" (Medicprise LP). The choice of suitable contrast medium 
is governed by the principles set forth above. 
One or more contrast media can be administered. The media may be 
administered as such or coupled to a targeting compound, such as a 
monoclonal antibody or peptide, that is able to attach to a surface 
protein specific to the maternal placenta. 
The contrast medium can be administered by injection into the maternal 
circulatory system, for example, in arteries or veins or by any other mode 
that selectively targets the maternal placenta. Alternatively, the 
contrast medium can be instilled into the amniotic cavity or injected 
directly into the fetal circulation, for example, by fetal cordo-infusion. 
The amount of the contrast agent administered can vary widely, depending on 
the type of contrast agent used, the route of administration, the desired 
bloom intensity or duration, and the possibility of any adverse effects to 
the mother or fetus. Typically, the dosage level is somewhere in the range 
of about 0.5 to about 20 cc/kg, preferably about 0.1 to about 10 cc/kg, 
most preferably from about 0.2 to about 5 cc/kg. 
The claimed invention allows direct visualization of movement in the 
arcuate arteries, spiral arteries and intervillous space, thus 
ascertaining the level of maternal blood flow into the placenta and 
evaluating whether the pregnancy is progressing normally. Previously, this 
aspect of maternal circulation has not been visible without the need to 
interfere with fetal circulation. 
Further, when high quality visual images representing the ultrasound 
information are captured on high quality VHS video recording tape, 
subsequent videodensitometry analysis can be performed. An initial 
densitometric analysis of the placental flow can provide a time-intensity 
curve, which has potential to quantify the wash-out characteristics of the 
contrast medium. For example, FIGURE 1 demonstrates graphically the change 
in intensity versus time for two different compositions. "Density--E" in 
FIGURE 1 identifies the time intensity curve for the contrast medium 
galactose particles (sold by Schering AG, Berlin, Germany under the trade 
name Echovist.RTM.). "Density--S" in FIGURE 1 identifies the time 
intensity curve for agitated saline. 
Using one aspect of the claimed method, by recording the increase in 
perceptible blood flow after the injection of a standard contrast medium 
followed by its fading away, a graphic representation of "normal" 
placental blood flow activity can be established. Thus, by manipulating 
the chemical nature of the contrast medium to produce a "standard" 
time-intensity curve, "normal" placentation can be better understood and 
defined in terms of the minimally acceptable volume and velocity of 
maternal blood flow. Time-intensity curves which deviate significantly 
from the norm can then be used to identify a placenta with abnormalities 
or meaningful pathologies. 
For example, when significant abnormalities are present, one may not be 
able to achieve as intense a bloom peak, or to achieve a bloom peak in the 
usual time, perhaps not at all. Alternatively, a prolonged fade period may 
be a key characteristic of normal, healthy placentas. Thus, useful 
parameters that can for the first time be quantified reliably using the 
claimed method include peak intensity, the area under the time-intensity 
curve, bloom half-life, and other measurements arising from various types 
of algorithmic analyses. 
Therefore, the claimed invention provides a method for characterizing what 
"normal" placentation means and defining various pathological conditions 
in terms of measurable parameters. Further, this method of identifying 
placenta pathologies can be used to intervene with therapeutic treatment, 
if necessary, at an earlier time when treatment is likely to be more 
meaningful. For example, appropriate responses to a time-intensity curve 
exhibiting significant abnormalities might indicate that the mother's 
behavior should be modified to optimize blood flow to the placenta, for 
example, restrictions in the level of the mother's physical activities or 
nutritional habits. 
The invention will be further clarified by the following example, which are 
intended to be purely illustrative of the invention. 
EXAMPLE 1 
Contrast Ultrasonography of Uteroplacental Blood Flow 
A study of nine pregnant female baboons was performed under medical and 
veterinary supervision. The baboons (P. cynocephalus) were each in the 
range of 6-12 years of age and each weighed in the range of 15-18 Kg. 
At 89 to 137 days of gestation, the pregnant baboons were each immobilized 
and maintained with 15 mg/kg of ketamine hydrochloride IM (sold by 
Parke-Davis under the trade name Ketalar.RTM.). Adequate tranquilization 
was provided for performing the sonography and to assure adequate blood 
pressure monitoring. A catheter was inserted into any accessible vein of 
each animal to collect blood for CBC and clinical chemistry. About 0.25 
mg/kg of diazepam (sold by Roche Products under the trade name Valium.RTM. 
IV) was injected, with exact doses varying as needed to keep the animal 
tranquilized and to establish baseline measurements. After an 18 gauge 
catheter was then inserted percutaneously into either the left or right 
femoral artery for blood gas sampling and monitoring, the blood pressure 
and pulse oximetry monitoring was begun. 
Prior and subsequent to injection of contrast media, a complete fetal 
biophysical profile was obtained. The contrast media were injected 
arterially or intravenously followed by a 5 cc saline flush. The contrast 
medium was injected while monitoring fetal EKG and separate, but 
simultaneous, ultrasound assessment of the arterial cardiac chamber. 
2-D gray-scale ultrasound and color Doppler were used to study 
uteroplacental and intervillous blood flow as follows. The ultrasound 
transducer was fixed on the animal's abdomen and focused on either a full 
cross-section of the placenta or an individual cotyledon (a section of the 
placenta). Once in a steady state, the gain of the transducer was then 
reduced to produce a low intensity signal, so that very little Doppler or 
color Doppler activity could be seen on the CRT visual display connected 
via a computer to the transducer. 
When the contrast medium was introduced, the flushing of the medium through 
the placenta could be observed as a lighting up of the intervillous space 
to form a "bloom." The bloom caused by the medium then faded away as the 
medium was washed out of the intervillous space. Various types of contrast 
media and degassed saline (control) were injected into the femoral 
arteries of the various animals tested, as needed. Ultrasound studies were 
repeated after each injection at standardized intervals. 
The contrast media tested were lipid coated microspheres (Filmix-Vet.RTM.), 
nitrogen-filled liposomes (Aerosomes.RTM.), galactose-encapsulated 
gas-filled bubbles (Echovist.RTM.), and agitated saline, with de-gassed 
saline being used as a control (no contrast medium). A summary of the 
gestation periods of the different animals and the various contrast media 
used with each is shown below: 
TABLE 1 
______________________________________ 
Animal Gestation Medium Dose Comments 
______________________________________ 
A 137 (78%) Aero- 0.3 cc/kg 
--; 
somes .RTM. 
0.6 cc/kg 
--; 
0.6 cc/kg 
--. 
B 136 (78%) Aero- 0.6 cc/kg 
--; 
somes .RTM. 
1.1 cc/kg 
color image. 
C 132 (75%) Filmix- 0.3 cc/kg 
--; 
Vet .RTM. 1.2 cc/kg 
Placental 
ring. 
D 132 (75%) Filmix- 0.6 cc/kg 
Placental 
Vet .RTM. ring; 
1.2 cc/kg 
--. 
E 137 (78%) Echovist .RTM. 
5 .times. 2 cc 
Intra- 
arterial. 
F 132 (75%) Echovist .RTM. 
5 .times. 2 cc 
Intra- 
arterial. 
G 109 (62%) Saline 2 cc 6 days behind 
in growth; 
Echovist .RTM. 
5 .times. 2 cc 
Intra- 
arterial. 
H 89 (51%) Aero- 0.6 cc/kg 
--; 
somes .RTM. 
0.6 cc/kg 
--. 
I 89 (51%) Aero- 0.9 cc/kg 
Hand held 
somes .RTM. (arcuate 
artery); 
0.2 cc/kg 
Kidney and 
heart images. 
______________________________________ 
The parameters measured, both with and without contrast media, included 
subjective enhancement of 2-D, gray-scale Doppler and color Doppler flow 
images. The study compared basal state subjective (see comments in Table 
1) and objective ultrasound/Doppler information for each of the injected 
substances. 
In the gray-scale mode, immediately after injecting the contrast medium, 
actual particulates were observed moving in the direction of the blood 
flow. In the color mode, the bloom was exhibited on a color CRT monitor as 
increased color activity in that area, with different colors indicating 
various blood velocities. 
Maternal and fetal effects (cardiopulmonary parameters) of the injections 
were also monitored, with fetal well-being being assessed throughout the 
procedure. The following were monitored: 
For Placental Stroma (Intervillous Space) 
2-D gray-scale (lower magnification, full placental ring), both Static and 
Real time; 2-D RES (Region Expansion Selection, section of placenta, e.g., 
a cotyledon of the placenta or a portion thereof), both Static and Real 
time; Standard Color with Variance Map; 
For Myometrium and Arcuate Artery 
2-D gray-scale, both Static and Real time; 2-D RES, both Static and Real 
time; Standard Color with Variance Map; 
The Following Vital Signs were Monitored 
Blood Pressure, Heart Rate, Respiration Rate, and Arterial Blood Gases. 
By visual inspection of the images produced by gray scale or color Doppler, 
the results showed the appearance of the bloom when contrast media were 
added. In addition, all studies were recorded on high-resolution video 
tape for more detailed analysis. 
The results showed that, once the gain was properly adjusted, most of the 
ultrasound contrast media tested could be used successfully to augment 
sonographic examination of the placenta in pregnant animals, even enabling 
the visualization of the maternal blood flow through the intervillous 
space of the placenta. Further, none of the animals or their fetuses 
appeared to suffer any adverse effects, either at the immediate conclusion 
of the test or one week later when biophysical profiles were again 
performed for three of the animals. Therefore, it was concluded that 
uteroplacental blood flow could be evaluated during pregnancy, thus 
enabling the possibility for early therapeutic intervention.