Premature birth is the leading cause of perinatal mortality and morbidity
worldwide. We have defined preterm labor as a syndrome and determined
that at least 25 percent of all preterm births are to women with sub-clinical
intrauterine infection. Moreover, we have provided evidence that a substantial
number of premature neonates are critically ill before birth and proposed
that the onset of premature labor has survival value in the context of
intrauterine infection. This year, we conducted a number of studies focusing
on the role of matrix-degrading enzymes in preterm premature rupture of
membranes (PROM). After premature birth, congenital anomalies are the
second leading cause of perinatal mortality in the United States. Advances
in imaging techniques have allowed the detection of many anatomical defects
before birth with ultrasound. The diagnostic power of three-dimensional
ultrasound is illustrated by the improved detection of cleft lip, vasa
previa, and congenital anomalies. This year, we reported the use of three-dimensional
sonography in the prenatal diagnosis of micrognathia, absence of the nasal
bone during the second and third trimester with trisomy 21, and the use
of color power imaging to map fetal vascular anatomy.
Role for Matrix Metalloproteinase in Spontaneous
Rupture of the Fetal Membranes
Chaiworapongsa, Edwin, Kim, Espinoza, Romero
The fetal membranes normally rupture after the onset of spontaneous labor.
PROM occurs in 10 percent of women at term and is associated with 30 to
40 percent of preterm deliveries. The mechanisms responsible for PROM
remain poorly understood. Histologic studies of the site of rupture have
demonstrated a zone of altered morphology characterized by thickening
of the connective tissue components of the membranes and thinning of the
cytotrophoblast layer and decidua as well as disruption of the interconnection
between amnion and chorion. A significant decrease in the density of collagen
type I, III, and V has been reported in the zone of altered morphology,
which is in close proximity to the cervix. Increased intrauterine pressure
during labor is thought to exert pressure over this area of the membranes,
resulting in membrane rupture. Thus, the current hypothesis is that increased
protease activity leads to an accelerated degradation of the extracellular
matrix of the fetal membranes and that this degradation, coupled with
biomechanical forces associated with labor, eventually leads to membrane
rupture. In the context of intrauterine infection, proteases produced
by bacteria or host cells could decrease membrane strength and elasticity.
This year, we conducted studies on the role of matrix metalloproteinase
3 (MMP-3), also known as stromelysin 1, in preterm PROM and microbial
invasion of the amniotic cavity; we also conducted studies on the role
of neutrophil elastase and secretory leukocyte protease inhibitor. Importantly,
we investigated whether the fetal carriage of single nucleotide polymorphisms
for MMP-1 and MMP-9 is associated with preterm PROM.
Neutrophil Elastase and MMP-3 in Preterm PROM
Chaiworapongsa, Espinoza, Edwin, Romero
Neutrophil elastase, a multifunctional serine protease stored in azurophilic
granules of mature neutrophils, is capable of intracellular degradation
of proteins during phagocytosis and extracellular degradation of connective
tissue during an inflammatory process. Secretory leukocyte protease inhibitor
(SLPI) is a natural neutrophil elastase inhibitor present in amniotic
fluid, fetal membranes, and cervical mucus. An imbalance between neutrophil
elastase and SLPI has been implicated as a mechanism of abnormal tissue
destruction in chronic inflammatory diseases, and we propose that it may
be involved in the mechanisms of membrane rupture. A series of studies
indicated that preterm PROM was associated with a significant increase
in the amniotic fluid concentration of neutrophil elastase and that microbial
invasion of the amniotic cavity was associated with a significant increase
in the amniotic fluid concentration of neutrophil elastase in women with
preterm labor and intact membranes as well as in women with preterm PROM.
Moreover, in the absence of microbial invasion of the amniotic cavity,
preterm and term PROM were associated with a significant reduction in
the amniotic fluid concentration of SLPI. These observations add strength
to the concept that intra-amniotic inflammation and host proteases participate
in the mechanism of membrane rupture.
Stromelysin 1 (MMP-3), produced in the context of infection, is able
to activate the latent forms of other MMPs. In contrast to the observations
that implicate neutrophil collagenase in the mechanism of membrane rupture,
preterm PROM was not associated with an increase in MMP-3 concentrations
in the amniotic fluid, although its concentrations increased in the context
of intra-amniotic infection.
Association of Genetic Predisposition to Preterm
PROM with Single Nucleotide Polymorphisms for MMP-1 and MMP-9
Romero
Our previous studies have provided evidence for the participation of MMP-9
and MMP-1 in preterm PROM. Recent studies have reported functional polymorphisms
for genes encoding these enzymes. We conducted two studies to determine
if these polymorphisms would raise the expression of MMP-1 and MMP-9 by
amnion cells and whether their frequency may be elevated in patients with
preterm PROM. A polymorphism at -1607 in the MMP-1 promoter (an insertion
of a guanine, G) creates a core Ets-binding site and increases promoter
activity. The 2G promoter was more than twice as active as the 1G allele
in amnion mesenchymal cells and a clone amnion cell line. Phorbol 12-myristate
13-acetate (PMA) increased mesenchymal cell nuclear protein binding with
greater affinity to the 2G allele. Induction of MMP-1 mRNA by PMA was
significantly greater in cells with a 1G/2G or 2G/2G genotype compared
with cells homozygous for the 1G allele. When treated with PMA, the 1G/2G
and 2G/2G cells produced greater amounts of MMP-1 protein than 1G/1G cells.
We found a significant association between fetal carriage of a 2G allele
and preterm PROM. Thus, the 2G allele has stronger promoter activity in
amnion cells and confers increased responsiveness of amnion cells to stimuli
that induce MMP-1; carriage of this polymorphism increases the risk of
preterm PROM.
We studied the functional significance of a variable-number tandem repeat
and a single nucleotide polymorphism (SNP) in the MMP-9 gene on promoter
activity and their association with preterm PROM. The 14 CA-repeat allele
was a stronger promoter than the 20 CA-repeat allele in amnion epithelial
cells and WISH amnionderived cells, but in THP-1 monocyte/macrophage
cells the 14 and 20 CA-repeat alleles exhibited similar activities. An
SNP at -1562 did not significantly affect promoter activity. A case-control
study indicated that the 14 CA-repeat allele was more common in newborns
delivered to mothers with preterm PROM than in those delivered at term.
There was no association between the -1562 SNP and preterm PROM. Thus,
the evidence suggests that cell hostdependent differences in MMP-9
promoter activity are related to the CA-repeat number and that fetal carriage
of the 14 CA-repeat allele is also associated with preterm PROM.
Three-Dimensional Ultrasonographic Presen-tation
of Micrognathia
Chaiworapongsa, Kalache, Romero
Micrognathia refers to a facial malformation characterized by a small
chin. The National Library of Medicines Online Mendelian Inheritance
in Man database includes 211 genetic conditions with such a malformation.
Affected fetuses are at increased risk for an abnormal karyotype. An underdeveloped
chin may be severe enough to cause polyhydramnios or respiratory obstruction
after delivery. Micrognathia can complicate infant feeding and typically
requires surgical repair. The prenatal diagnosis of micrognathia is difficult
and largely subjective. We have used three-dimensional ultrasound to examine
the fetal mandible and developed an approach for the diagnosis. We used
orthogonal multiplanar views to obtain a midsagittal facial profile. The
most important advantage of three-dimensional ultrasound is its ability
to display a true midsagittal plane of the fetal face. Diagnostic inaccuracies,
however, can result from inappropriate manipulation of multiplanar images
and rendered volume data. One of the advantages of three-dimensional ultrasound
surface rendering is that it enables referring physicians to counsel prenatal
patients accurately.
Evaluation of Spina Bifida by Three-Dimensional
Ultrasonography
Chaiworapongsa, Romero
The most common and difficult-to-diagnose neural tube defect is spina
bifida. Advances in treatment have greatly reduced mortality, but they
have had minimal impact on long-term disability, which includes paraplegia,
sensory deficits, spinal deformity, bowel dysfunction, and urinary incontinence.
Prenatal ultrasonography can detect between 80 to 85 percent of fetuses
with spina bifida. The location and extent of the lesion are critical
factors in assessing the likelihood of long-term disability. By using
two- and three-dimensional ultrasonography to determine the extent of
vertebral defects among affected fetuses, we have developed a novel approach
for the systematic examination of the fetal spine and the mapping of defects.
We counted spinal levels independently from the most caudal thoracic vertebra
with a rib (e.g., 12th thoracic rib) and manipulated a virtual cutting
plane through a volume-rendered spine to generate optimal multiplanar
views for this blinded analysis. We compared prenatal diagnosis with a
postnatal analysis of bony spine defects derived from radiographic films
or magnetic resonance imaging and used two-dimensional and three-dimensional
ultrasonography to examine fetuses. For two-dimensional ultrasonography,
the spinal level agreed to within one vertebral segment in six of nine
infants. In contrast, three-dimensional ultrasonography agreed to within
one vertebral segment in eight of nine infants. Three fetuses had vertebral
defect levels on two-dimensional ultrasonography that were 1.5 to two
segments away from postnatal findings. The same fetuses had results that
were within one vertebral segment on three-dimensional ultrasonography.
Volume rendering showed splayed vertebral pedicles and disrupted vertebrae.
An intact meningeal sac was easily rendered in five of nine subjects.
Multiplanar views were more informative than rendered views for localizing
bony defects of the fetal spine.
Nasal Bone Evaluation in Fetuses with Down Syndrome
during the Second and Third Trimesters of Pregnancy
Kalache, Chaiworapongsa, Romero
The standard method for the prenatal diagnosis of trisomy 21 is karyotype
of cells obtained through amniocentesis, a procedure with a 0.5 percent
risk of pregnancy loss. In response to considerable interest in the identification
of sonographic markers as an alternative diagnostic tool, recent studies
suggest that a non-visualized nasal bone may identify fetuses with trisomy
21 in the first trimester of pregnancy. Yet, little information is available
on the diagnostic potential of this sonographic finding in the second
and third trimesters of pregnancy, which is when most sonograms are performed
in the United States. Three-dimensional ultrasound uses multiplanar views
that allow for standardization of the plane for the examination of the
fetal nasal bone. We conducted a study to determine the value of this
technology in assessing the presence or absence of the fetal nasal bone
in normal fetuses as well as in those with trisomy 21 and found that three-dimensional
ultrasound allowed substantial agreement in scoring whether or not the
nasal bone was visualized. Forty percent of fetuses with trisomy 21 had
absence of the nasal bone. In contrast, we identified the nasal bone in
80 to 90 percent of normal fetuses. Our most important finding was that
three-dimensional ultrasound allowed standardization of the diagnosis.
Genetic Sonography, a Cost-Effective Method for
Evaluating Women 35 Years and Older Who Decline Genetic Amniocentesis
Romero
Given that the frequency of aneuploidy increases with advancing maternal
age, standard clinical practice calls for offering invasive testing (chorionic
villus sampling or second-trimester amniocentesis) to women 35 years of
age and older. Following genetic counseling, not all women elect invasive
testing. Some patients inquire whether ultrasound examination of the fetus
would provide additional information regarding their risk for aneuploidy
or other genetic diseases. Accordingly, the sonographer or sonologist
must be able to identify fetal ultrasound features associated with aneuploidy.
Previous studies have indicated that the sensitivity of genetic ultrasound
for detection of a fetus with trisomy 21 ranges from 25 to 91 percent
and that the sensitivity for detecting all chromosomal abnormalities in
women of advanced maternal age is 81 percent. In addition, an incremental
cost is associated with genetic ultrasound. We conducted a study to determine
if offering genetic ultrasound to patients who decline invasive testing
is cost-effective and can increase the detection rate of trisomy 21. We
determined the detection rate of trisomy 21, the number of pregnancy losses
after amniocentesis, and the cost of detecting a single fetus with trisomy
21 in women 35 years and older. We concluded that offering genetic sonography
followed by amniocentesis to patients 35 years and older who originally
decline invasive testing for the diagnosis of trisomy 21 is cost-effective
and results in a higher overall detection rate for trisomy 21 without
an increased risk of pregnancy loss.
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SELECTED PUBLICATIONS
- Chaiworapongsa T, Gervasi MT, Refuerzo J, Espinoza J, Yoshimatsu
J, Berman S, Romero R. Maternal lymphocyte subpopulations (CD45RA+ and
CD45RO+) in preeclampsia. Am J Obstet Gynecol. 2002;187:889-893.
- Chaiworapongsa T, Romero R, Kim JC, Kim YM, Blackwell SC, Yoon BH,
Gomez R. Evidence for fetal involvement in the pathologic process of
clinical chorioamnionitis. Am J Obstet Gynecol. 2002;186:1178-1182.
- Chaiworapongsa T, Romero R, Yoshimatsu J, Espinoza J, Kim YM, Park
K, Kalache K, Edwin S, Bujold E, Gomez R. Soluble adhesion molecule
profile in normal pregnancy and pre-eclampsia. J Matern Fetal Neonatal
Med. 2002;12:19-27.
- DeVore GR, Romero R. Genetic sonography: a cost-effective method
for evaluating women 35 years and older who decline genetic amniocentesis.
J Ultrasound Med. 2002; 21:5-13.
- Ferrand PE, Parry S, Sammel M, Macones GA, Kuivaniemi H, Romero R,
Strauss JF III. A polymorphism in the matrix metalloproteinase-9 promoter
is associated with increased risk of preterm premature rupture of membranes
in African Americans. Mol Hum Reprod. 2002;8:494-501.
- Fujimoto T, Parry S, Urbanek M, Sammel M, Macones G, Kuivaniemi H,
Romero R, Strauss JF. A single nucleotide polymorphism in the matrix
metalloproteinase-1 (MMP-1) promoter influences amnion cell MMP-1 expression
and risk for preterm premature rupture of the fetal membranes. J Biol
Chem. 2002;277:6296-6302.
- Helmig RB, Romero R, Espinoza J, Chaiworapongsa T, Bujold E, Gomez
R, Ohlsson K, Uldbjerg N. Neutrophil elastase and secretory leukocyte
protease inhibitor in prelabor rupture of membranes, parturition and
intra-amniotic infection. J Matern Fetal Neonat Med. 2002;12:237-246.
- Lee W, Chaiworapongsa T, Romero R, Williams R, McNie B, Johnson A,
Treadwell M, Comstock CH. A diagnostic approach for the evaluation of
spina bifida by three-dimensional ultrasonography. J Ultrasound Med.
2002; 21:619-626
- Lee W, McNie B, Chaiworapongsa T, Conoscenti G, Kalache KD, Vettraino
IM, Romero R, Comstock CH. Three-dimensional ultrasonographic presentation
of micrognathia. J Ultrasound Med. 2002; 21:775-781.
- Romero R, Espinoza J, Chaiworapongsa T, Kalache K. Infection and
prematurity and the role of preventive strategies. Semin Neonatol. 2002;7:259-274.
COLLABORATORS
Greggory DeVore, M.D., ALFIGEN-The Genetics Institute,
Pasadena, CA
Helena Kuivaniemi, Wayne State University School
of Medicine, Detroit, MI
Wesley Lee, M.D., William. Beaumont Hospital,
Royal Oak, MI
George A. Macones, M.D., University of Pennsylvania
Medical Center, Philadelphia, PA
Jerome F. Strauss, M.D., Ph.D., University of
Pennsylvania Medical Center, Philadelphia, PA
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