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 amnion–derived 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 host–dependent 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 Medicine’s 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.