PATHOPHYSIOLOGY OF PREMATURE LABOR AND COMPLICATIONS OF
PREMATURITY;
PRENATAL DIAGNOSIS OF CONGENITAL ANOMALIES
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Roberto
Romero, MD, Unit on Maternal Fetal
Medicine, Pathology Unit, Parturition Unit Samuel
Edwin, PhD, Senior Research Assistant Joon-Seok
Hong, MD, Visiting Fellow Tinnakorn
Chaiworapongsa, MD, Postdoctoral Fellow Jimmy
Espinoza, MD, Postdoctoral Fellow Gi
Jin Kim, PhD, Postdoctoral Fellow Mi Ran
Kim, DVM, Postdoctoral Fellow Jyh
Kae Nien, MD, Postdoctoral Fellow |
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Premature
birth is the leading cause of perinatal mortality and morbidity worldwide. We
have defined pre-term labor as a syndrome and determined that at least 25 percent
of pre-term neonates are born to women with subclinical intrauterine
infection. Moreover, we have provided evidence that many premature neonates
are critically ill before birth and proposed that, in the context of
intrauterine infection, the onset of premature labor has survival value. One
focus of our research is to understand the pathophysiology of premature labor
and delivery by studying several components of the innate immune system,
including neutrophils and Toll-like receptors as well as the role of allergy
in causing pre-term labor and delivery. We also used a genome-wide approach
to identify differentially regulated genes in patients with premature rupture
of membranes. The second focus of our research is to improve the diagnosis
and treatment of fetal disease and congenital anomalies, which are the second
leading cause of perinatal mortality in the Association
of pregnancy with changes in the adaptive and innate immune response Chaiworapongsa,
Espinoza, Edwin, Romero Although
some investigators have proposed that pregnancy is an
“anti-inflammatory state” thought to be beneficial for survival
of the fetal semi-allograft, others have suggested that pregnancy is
characterized by “activation of the innate immune system” to
compensate for impaired adaptive immunity and to protect the mother from
infection. We made the following observations when studying (1) neutrophils
and monocytes of normal pregnant women and those with complications and (2)
the cell biology that underlies metabolic changes in these cells. Phenotypic
and metabolic profile of monocytes and granulocytes in normal pregnancy:
Granulocytes from normal pregnant women overexpress CD14 and CD64 when
compared with granulocytes of nonpregnant women. The baseline intracellular
reactive oxygen species, oxidative burst, and stimulation index values were
higher in the granulocytes of normal pregnant women than in those of
nonpregnant women. However, phenotypic and metabolic changes were more marked
in acute systemic maternal infection, indicating that the innate limb of the
immune response is not maximally stimulated during normal pregnancy. Phenotypic
and metabolic profile of monocytes and granulocytes in preeclampsia, pre-term
labor, and pre-term premature rupture of membranes: Flow
cytometry studies indicated that these three complications of pregnancy are
associated with phenotypic and metabolic changes consistent with activation. Controversy
about impairment of neutrophil and macrophage function during pregnancy: In
vitro studies suggest that many neutrophil functions are depressed in
normal pregnancy, including microbial killing, chemotaxis, phagocytosis, and
adhesion. Yet, the results appear to conflict with those reported by recent
investigations. We conducted several studies to explore the reason for the
conflicting results. Oxidant
production by neutrophils is dependent on NADPH oxidase, which can be
regulated by mechanisms related to enzyme assembly or substrate-level
regulation. The effects of different factors, including cytokines, are
collectively expressed as NADPH metabolic oscillations, which can be
characterized by defining their amplitude and frequency. We performed our
studies by using nonperturbative quantitative microfluorometry, which allows
real-time single-cell assay of reactive oxygen radical production. We found
that neutrophils from normal pregnant women have an increased number of
high-amplitude oscillations (20 seconds); an even higher percentage of cells
derived from patients suffering from maternal infection and preeclampsia
display the same type of oscillations. However, in other complications of
pregnancy, the neutrophil metabolic profile showed a reduced number of
high-amplitude oscillations. We
found that, during pregnancy, even though neutrophils displayed
high-amplitude NADPH oscillations, they lose the ability to change (increase)
their frequency, a state that is unique to pregnant women. We have identified
a novel and fundamental mechanism for oxidant regulation, which accounts for
the higher basal activity but the inability to “activate”
pregnancy neutrophils. Translocation of enzymes in the hexose monophosphate
shunt accounts for reduced production of reactive oxygen metabolites. This
model resolves discrepancies in the literature, and studies are in progress
to define the mechanism responsible for both amplitude and frequency
modulation of NADPH oscillations. Induction
of premature labor and delivery by allergic reaction and prevention by
histamine H1 receptor antagonist Chaiworapongsa, Romero We
proposed that a subgroup of patients with premature labor may have an
allergic-like mechanism of disease responsible for pre-term birth. The
studies conducted this year provided evidence that sensitization of animals
and subsequent challenge during pregnancy is able to cause premature labor
and delivery. At days 40 through 50 of gestation, ovalbumin-sensitized guinea
pigs that had not been treated or had been pretreated with an H1 receptor
antagonist (ketotifen) or a mast-cell stabilizer (cromolyn sodium) were
challenged with either ovalbumin or saline solution. Nonsensitized guinea
pigs were challenged with ovalbumin. The duration of gestation was
significantly shorter in sensitized animals challenged with ovalbumin than in
the other groups. Pretreatment with ketotifen significantly increased the
duration of pregnancy in sensitized animals as compared with untreated
animals, whereas cromolyn sodium had no effect. Our observations support the
concept that a Type-1 hypersensitivity reaction is a mechanism of disease in
premature labor, which is important given that an estimated 40 to 60 million
Americans have allergic disorders. Deficient
expression of proteinase inhibitor 3 in premature rupture of membranes
revealed by genome-wide expression profiling of fetal membranes Chaiworapongsa, Kim M,
Kim G, Espinoza, Edwin, Romero Pre-term
premature rupture of membranes (PPROM) is a leading cause of pre-term birth.
A growing body of evidence supports the concept that pre-term parturition is
a syndrome with multiple causes that is defined by the presence of increased
uterine contractility, cervical dilatation, and decidual/membrane activation
with or without PROM. One approach to dissect the mechanisms of disease in
PPROM is genomics. We conducted a study to determine mRNA expression profiles
with microarrays to classify pre-term birth with intact membranes and PPROM
in the presence and absence of acute chorioamnionitis. Microarray experiments
identified the decreased expression of proteinase inhibitor 3 (PI3) in the
PPROM cases, results that we confirmed by quantitative reverse
transcriptase-polymerase chain reaction. Immunohistochemistry demonstrated
decreased PI3 protein expression in PPROM. The significance of the finding is
that the protein had not been previously implicated in parturition and,
specifically, in membrane rupture. Our observations suggest a novel pathway,
which should be investigated, in the pathobiologic condition of pre-term
birth. PI3 is a low–molecular weight serine proteinase inhibitor. We
propose that the production of PI3 in the fetal membranes protects the tissue
from damage that could be caused by increased activity and availability of
neutrophil elastase. Patients incapable of producing adequate amounts of PI3
may be predisposed to PPROM. Bytautiene E, Romero R, Vedernikov Y, El-Zeky F, Saade G,
Garfield R. Induction of premature labor and delivery by allergic reaction
and prevention by histamine H1 receptor antagonist. Am J Obstet Gynecol 2004;
191:1136-1161. Chaiworapongsa T, Romero R, Espinoza Dorado J, Bujold E, Kim YM,
Gomez R, Edwin SS. Evidence supporting a role for blockade of the vascular
endothelial growth factor system in the pathophysiology of preeclampsia.
Young Investigator Award. Am J Obstet Gynecol 2004;190:1547-1550. Kim YM, Romero R, Chaiworapongsa T, Kim GJ, Kim M, Kuivaniemi
SH, Tromp G, Espinoza Dorado J, Bujold E, Abrahams VM, Mor G. Toll-like
receptor-2 and -4 in the chorioamniotic membranes in spontaneous labor at
term and in preterm parturition associated with chorioamnionitis. Am J
Obstet Gynecol 2004;191:1346-1355. Kindzelskii AL, Ueki T, Michibata H, Chaiworapongsa T, Romero R,
Petty H. 6-phosphogluconate dehydrogenase and glucose-6-phosphate
dehydrogenase form a supramolecular complex in human neutrophils that
undergoes retrograde trafficking during pregnancy. J Immunol
2004;172:6373-6381. Tromp G, Kuivaniemi SH, Chaiworapongsa T, Kim YM, Maymon E,
Edwin SS, Romero R. Genome-wide expression profiling of fetal membranes
reveals a deficient expression of proteinase inhibitor 3 in premature rupture
of membranes. Am J Obstet Gynecol 2004;191:1331-1338. Four-dimensional
ultrasonography of the fetal heart using Spatio-Temporal Image Correlation Chaiworapongsa,
Espinoza, Romero We
described a new technique for the examination of the fetal heart. Using a new
cardiac gating technique, which uses automated transverse and longitudinal
sweeps of the anterior chest wall, we acquired volume data sets of the fetal
heart. The volumes were obtained from 69 fetuses: 35 normal, 16 with
congenital anomalies not affecting the cardiovascular system, and 18 with
cardiac abnormalities. We performed dynamic multiplanar slicing and surface
rendering of cardiac structures. To illustrate the Spatio-Temporal Image
Correlation (STIC) technique, we compared two representative volumes from a
normal fetus with volumes obtained from fetuses with the following congenital
heart anomalies: atrioventricular septal defect, tricuspid stenosis,
tricuspid atresia, and interrupted inferior vena cava with abnormal venous
drainage. We used volume data sets obtained with a transverse sweep to
demonstrate cardiac anatomy. Using a reference dot to navigate the
four-chamber view, we could simultaneously study intracardiac structures in
three orthogonal planes and interactively manipulate volumes to visualize
both outflow tracts simultaneously in addition to the aortic and ductal
arches, thereby generating novel views of specific structures. Dynamic
multiplanar slicing and surface rendering of the fetal heart are thus
feasible with STIC technology. One good-quality volume data set, obtained
from a transverse sweep, can be used to examine the four-chamber view and the
outflow tracts. This novel methodology may assist in the evaluation of fetal
cardiac anatomy. We also described the use of color Doppler STIC, which
allowed multiplanar visualization of ventricular septal defects, multiplanar
display and volume rendering of tricuspid regurgitation, volume rendering of
the outflow tracts by color and power Doppler ultrasonography (both in a
normal case and in a case of a double-inlet right ventricle with a
double-outlet right ventricle), and visualization of venous streams at the
level of the foramen ovale. This technique has the potential to simplify
visualization of the outflow tracts and to improve the evaluation of the
location and extent of ventricular septal defects. Other applications include
three-dimensional evaluation of regurgitation jets and venous streams at the
level of the foramen ovale. Espinoza J, Goncalves LF, Lee W, Chaiworapongsa T, Treadwell MC,
Stites S, Schoen ML, Mazor M, Romero R. The use of the minimum projection
mode in 4-dimensional examination of the fetal heart with spatiotemporal
image correlation. J Ultrasound Med 2004;23:1337-1348. Espinoza J, Kalache K, Goncalves LF, Lee W, Chaiworapongsa T,
Schoen ML, Devers P, Treadwell M, Mazor M, Romero R. Prenatal diagnosis of
membranous ventricular septal aneurysms and their association with absence of
atrioventricular valve ‘offsetting.’ Ultrasound Obstet Gynecol
2004;24:787-792 Agreement
between observers and reproducibility of a technique to display standard
cardiac views of the left and right ventricular outflow tracts from
four-dimensional volume data sets acquired with Spatio-Temporal Image
Correlation Chaiworapongsa,
Espinoza, Romero We
developed a technique to obtain dynamic multiplanar images of the left
ventricular outflow tract (LVOT) and right ventricular outflow tract (RVOT)
from volume data sets acquired with STIC. We obtained volume data sets from
fetuses with normal cardiac anatomy. One investigator selected 20 volume data
sets of satisfactory quality. The data were randomly assigned for a blind
review by two independent observers with previous experience in fetal
echocardiography. One volume data set only was used for each fetus. After a
training session, the observers obtained standardized cardiac views of the
LVOT and RVOT, which were scored on a scale of 1 to 5 based on diagnostic
value and image quality (1 = unacceptable, 2 = marginal, 3 = acceptable, 4 =
good, and 5 = excellent). We calculated median scores and interquartile
range, as well as interobserver and intraobserver agreement, for each view.
Median scores (interquartile range) for LVOT images, obtained by the first
and second observers, were 3.5 (2.25 to 5.00) and 4 (3.00 to 5.00),
respectively. The median scores (interquartile range) for RVOT images
obtained by the first and second observers were 3 (3.00 to 5.00) and 3 (2.00
to 4.00), respectively. The interobserver intraclass correlation coefficient
for the LVOT was 0.693 (95 percent CI 0.380 to 0.822), and 0.696 (95 percent
CI 0.382 to 0.866) for the RVOT. For the intraobserver agreement analysis,
observer 1 gave higher scores to the LVOT the second time the volumes were
analyzed [LVOT: 3.50 (2.25 to 5.00) versus 5.00 (4.00 to 5.00, p=0.008)].
Therefore, STIC can be reproducibly used to evaluate fetal cardiac outflow
tracts by independent examiners. Slightly better image quality rating scores
during the intraobserver variability trial suggest the need for operator
training for the manipulation and analysis of volume data obtained by STIC. Goncalves LF, Espinoza J, Mazor M, Romero R. Newer imaging
modalities in the prenatal diagnosis of skeletal dysplasias. Ultrasound
Obstet Gynecol 2004;24:115-120. Goncalves LF, Espinoza J, Romero R, Lee W, Beyer B, Treadwell
MC, Humes R. A systematic approach to prenatal diagnosis of transposition of
the great arteries using 4-dimensional ultrasonography with spatiotemporal
image correlation. J Ultrasound Med 2004;23:1225-1231. Goncalves LF, Lee W, Chaiworapongsa T, Espinoza J, Schoen ML,
Falkensammer P, Treadwell M, Romero R. Four-dimensional ultrasonography of
the fetal heart with spatiotemporal image correlation. Am J Obstet Gynecol
2003;189:1792-1802. Goncalves LF, Romero R, Espinoza J, Lee W, Treadwell M, Chintala
K, Brandl H, Chaiworapongsa T. Four-dimensional ultrasonography of the fetal
heart using color Doppler spatiotemporal image correlation. J Ultrasound
Med 2004;23:473-481. Lee W, Deter RL, McNie B, Goncalves LF, Espinoza Dorado J,
Chaiworapongsa T, Romero R. Individualized growth assessment of fetal soft
tissue using fractional thigh volume. Ultrasound Obstet Gynecol
2004;24:766-774. COLLABORATORS Robert Garfield, MD, Luis F. Goncalves, MD, Helena Kuivaniemi, MD, PhD, Wesley Lee, MD, Howard Petty, PhD, Gerard Tromp, PhD,
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