Roberto Romero, MD, Chief, Perinatology Research Branch
Samuel Edwin, PhD, Senior Research Assistant
Jyh Kae Nien, MD, Postdoctoral Fellow

We conduct clinical and laboratory research in maternal and fetal diseases responsible for excessive infant mortality in the United States. We study the mechanisms of disease responsible for preterm delivery, with particular emphasis on the role of maternal and fetal inflammation and subclinical infection. The prenatal diagnosis of congenital anomalies is also a major area of interest. Preterm birth is the leading cause of perinatal mortality and morbidity worldwide. We defined preterm labor as a syndrome and determined that at least 25 percent of preterm neonates are born to women with subclinical intrauterine infection. We also 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. Our goal is to understand the pathophysiology of premature labor and delivery. Congenital anomalies are the second leading causes of perinatal mortality in the United States after premature birth. Advances in imaging techniques have allowed the in utero detection of many anatomical defects with ultrasound, promising to improve the diagnosis and treatment of fetal disease and congenital anomalies.

Gene expression signature of spontaneous term labor without histologic chorioamnionitis

Romero

Human parturition involves ''a common pathway'' manifested clinically by uterine contractions, cervical ripening, and chorioamniotic membrane/decidual activation, culminating in membrane rupture. Previous studies have linked parturition to an inflammatory process. Using an unbiased genome-wide approach, we analyzed both the transcriptome in the chorioamniotic membranes and maternal blood in order to identify the biological processes in normal spontaneous labor. We generated transcriptional profiles for chorioamniotic membranes and blood from patients at term with no labor and from patients at term in labor. We subjected all placentas to histologic examination and used the absence of histologic chorioamnionitis as a criterion for inclusion. Among patients in term labor, we observed increased expression of several chemokines and transcripts associated with neutrophil and monocyte recruitment. The results of our study indicate that, despite the absence of histological chorioamnionitis, labor induces gene expression changes consistent with localized inflammation.

Uterine transcriptomes of bacteria-induced and ovariectomy-induced preterm labor in mice are characterized by differential expression of arachidonate metabolism genes

Romero

Preterm labor (PTL) is a syndrome with several etiologies. We focused on two well-established mechanisms of PTL: intrauterine infection and the requirement of progesterone for pregnancy maintenance. To investigate the role of intrauterine infection and progesterone in human PTL, we used two models to analyze the uterine transcriptome of mice: the first model was the induction of PTL by intrauterine infection with Escherichia coli, and the second model was the induction of PTL by ovariectomy and progesterone withdrawal. Quantitative reverse transcriptase-polymerase chain reaction measurements demonstrated that bacteria-induced PTL substantially increased the expression of genes involved in prostaglandin synthesis. In contrast, ovariectomy-induced PTL increased the expression of genes involved in lipoxin, leukotriene, and hydroxyeicosatetraenoic acid synthesis. The results of the study indicated that bacteria-induced and ovariectomy-induced PTL each express a different balance of genes involved in prostaglandin synthesis and the synthesis of lipoxins, leukotrienes, and HETEs. This balance may be important to PTL and is amenable to experimental investigation.

The transcriptome of the uterine cervix before and after spontaneous term parturition

Romero, Edwin; incollaboration with Haddad, Hassan, Kim Y

Cervical ripening is one of the components of the common pathway for parturition. Therefore, determining the expression profile of genes involved in the ripening process is crucial for understanding the mechanisms leading to premature cervical ripening and preterm delivery. Our study provided an unbiased and comprehensive description of the changes in the cervical transcriptome before and after spontaneous term labor. We used Affymetrix microarrays to characterize the transcriptome of cervical tissue from patients at term but not in labor and after spontaneous labor. The results revealed that the cervical transcriptome of term patients who underwent labor is dramatically different from that of patients without labor, with 1,192 differentially expressed genes. Among the upregulated genes were those involved in neutrophil chemotaxis, apoptosis, extracellular matrix regulation, and steroid metabolism. Genes involved in neutrophil chemotaxis were upregulated in specimens from women after spontaneous labor, and real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) confirmed an increased expression of IL-8, IL-6, and vascular endothelial growth factor. The expression of Toll-like receptor (TLR)-3 and TLR-5 declined in patients after spontaneous labor, as shown by the microarray analysis and confirmed by qRT-PCR.

A rapid bedside test for the prediction of preterm delivery

Romero, Nien, Edwin; in collaboration with Erez, Espinoza, Gomez, Hassan, Kusanovic, Richani, Soto

Intra-amniotic infection (IAI) and inflammation have been causally linked to PTL, preterm delivery (PTD), and fetal injury. Intra-amniotic inflammation can be detected by amniocentesis and cytokine measurements (e.g., IL-6 and MMP-8), white blood cell count, and glucose concentrations in the amniotic fluid. It has been previously demonstrated, with 95 percent sensitivity and 93 percent specificity, that elevated MMP-8 concentrations in the amniotic fluid of patients with PTL and intact membranes can diagnose intra-amniotic inflammation. This year, we investigated the diagnostic indices, predictive values, efficiency, and likelihood ratios of the MMP-8 PTD Check Test for the detection of IAI, inflammation, spontaneous PTD, and severe neonatal morbidity among patients with increased uterine contractions and intact membranes. The MMP-8 PTD Check Test (SK Pharma Co., Ltd., Kyunggi-do, Korea) is a rapid bedside test (configured similarly to a rapid pregnancy test) designed to provide a quick bedside assessment for elevated concentrations of MMP in a small amount of amniotic fluid. A positive MMP-8 rapid test correctly predicted 70 percent of the spontaneous PTDs occurring within 48 hours and 94 percent of those occurring within 7 to 14 days of the amniocentesis. The efficiency of a positive MMP-8 rapid test in the identification of IAI and inflammation was 94 percent (311/331) and 93 percent (308/331), respectively. We concluded that the rapid test provides clinicians with a fast and accurate assessment of the inflammatory status of the amniotic cavity, allowing for better identification of patients at risk for impending PTD. Follow-up clinical trials are required to determine the utility of the MMP-8 rapid test for the identification of inflammation at the time of amniocentesis and to determine whether, based on the rapid test results, treatment with antibiotics and/or anti-inflammatory agents may improve pregnancy outcome.

Phylogenetic analysis reveals the evolution of the mammalian placenta

Romero; incollaboration with Erez, Wildman

The placenta is the lifeline for mammalian reproduction and a complex organ that provides clues about natural selection and evolution. Through phylogenetic analysis of molecular and anatomical data, we obtained evidence describing the evolutionary history of the placenta of eutherian mammals, the group that includes all mammal species except marsupials and the egg-laying monotremes (for example, the duck-billed platypus). The study found, in contrast to most theories, that the disc-shaped, hemochorial placenta of many primates, including humans, existed throughout the eutherian lineage from the last common ancestor of placental mammals to the emergence of humans. That intimate contact between fetal and maternal blood was established in the last common ancestor of the crown group of Eutheria gives credence to the hypothesis that successful pregnancy requires appropriate allorecognition and tolerance at the maternal-fetal interface.

Tomographic ultrasound imaging for examination of the fetal heart

Romero, Nien; in collaboratin with Erez, Espinoza, Goncalves, Kusanovic, Soto

Using three-dimensional ultrasound, we initiated a series of projects to improve the detection of congenital anomalies and to assess fetal growth and development. We have invested substantial effort in the development of techniques to improve the prenatal diagnosis of congenital heart disease, which is the leading cause of death among fetuses with congenital anomalies.

A recent technological development allows automatic slicing of volume data sets acquired with three-dimensional or four-dimensional ultrasonographic techniques in the transverse, sagittal, or coronal planes. The technique allowed us to obtain a series of slices resembling the display commonly used to read computerized tomography or magnetic resonance. We sought to determine the feasibility of conducting a comprehensive echocardiographic examination in fetuses with and without congenital heart disease. Using the automated tomographic slicing of the fetal heart, we visualized standard views such as the four-chamber view, five-chamber view, and three-vessel and trachea view in 97.4 percent, 88.2 percent, and 79.5 percent of cases, respectively. The addition of color Doppler allowed visualization of these planes in 98.2 percent, 97.0 percent, and 83.6 percent of cases, respectively.

A novel algorithm for comprehensive fetal echocardiography using four-dimensional ultrasonography and tomographic imaging

Romero, Nien; in collaboratin with Espinoza, Goncalves, Hassan, Kusanovic, Lee

We explored the possibility of combining tomographic ultrasound imaging with a predefined set of manipulations of a volume data set in order to visualize systematically—in a single image divided into four frames—the four-chamber view, long axis view of the aorta, short axis view of the pulmonary artery, and three-vessel and trachea view of the fetal heart. The objectives of this novel algorithm are to facilitate the performance of a thorough fetal echocardiographic examination from a single volume data set of the fetal heart and to allow documentation of the essential planes of section in a single image in order to facilitate comparison. Use of the proposed algorithm made possible simultaneous visualization of the short axis of the pulmonary artery, three-vessel and trachea view, left outflow tract, and four-chamber view of the heart in 78 percent of the fetuses with no congenital heart disease and in 40 percent of those with congenital heart disease.

What does two-dimensional imaging add to three- and four-dimensional obstetric ultrasonography?

Romero, Nien; in collaboration with Espinoza, Goncalves, Kusanovic, Lee, Soto

When three-dimensional ultrasound is performed with mechanical probes, a volume data set is generated with precise spatial orientation by assembling a series of two-dimensional images obtained automatically by the probe. Three-dimensional volume data sets can then be sliced and displayed in any desired plane of section and should contain the same or more information than that provided by the original two-dimensional images. Investigators have recently explored the principle underlying assembly of the volume data set and have proposed that examination of volume data sets alone could be used to streamline ultrasound examinations in busy clinical practices. A crucial question is whether information contained in volume data sets is reliable. We conducted a blinded study comparing the diagnostic information provided by examination of volume data sets alone with information obtained during a conventional two-dimensional ultrasonographic examination. The same examiner, who was blinded to the indications for the examination, evaluated the three-dimensional volume data sets, established a diagnostic impression, and then conducted a two-dimensional ultrasound examination. Our innovative approach departs from earlier approaches in that previous studies first performed two-dimensional ultrasonography and then compared the results from two-dimensional with three-dimensional ultrasonography. Agreement between the three-dimensional and the two-dimensional ultrasound was observed for 90.4 percent of the findings, with no difference in sensitivity and specificity for the diagnosis of congenital anomalies between the two methods. In one case, artifacts related to fetal motion during acquisition led to an erroneous diagnosis of double-outlet right ventricle instead of transposition of the great arteries.

Applications of two-dimensional matrix array for three- and four-dimensional examination of the fetus

Romero, Nien; in collaboration with Espinoza, Goncalves, Kusanovic, Lee

We also explored the potential applications of two-dimensional matrix array technology for the examination of fetal structures other than the fetal heart. Two-dimensional matrix array is a new technology originally developed for the examination of adult hearts by real-time three-dimensional echocardiography. The advantage of the new technology is that it does not require reconstruction of two-dimensional images to form the volume data set; moreover, its temporal resolution is optimal. Our preliminary experience with this technology indicates that it allows 360-degree rotation of fetal structures in real time without moving the transducer, thereby facilitating examination of structures from several angles and potentially facilitating diagnosis of congenital anomalies. We identified limitations of currently available technology related to lower resolution and narrower volume display as compared with conventional three-dimensional volumetric techniques.

COLLABORATORS

Offer Erez, MD, Wayne State University School of Medicine, Detroit, MI
Jimmy Espinoza, MD, Wayne State University School of Medicine, Detroit, MI
Ricardo Gomez, MD, Sotero del Rio Hospital, Puente Alto, Chile
Luis F. Goncalves, MD, Wayne State University School of Medicine, Detroit, MI
Ramsi Haddad, PhD, Wayne State University School of Medicine, Detroit, MI
Sonia Hassan, MD, Wayne State University School of Medicine, Detroit, MI
Chong-Jai Kim, MD, PhD, Wayne State University School of Medicine, Detroit, MI
Jung Sun Kim, MD, PhD, Wayne State University School of Medicine, Detroit, MI
Yeon Mee Kim, MD, Wayne State University School of Medicine, Detroit, MI
Juan Pedro Kusanovic, MD, Wayne State University School of Medicine, Detroit, MI
Wesley Lee, MD, William Beaumont Hospital, Royal Oak, MI
Karina Richani, MD, Wayne State University School of Medicine, Detroit, MI
Eleazar Soto, MD, Wayne State University School of Medicine, Detroit, MI
Derek Wildman, MD, PhD, Wayne State University School of Medicine, Detroit, MI

For further information, contact warfiela@mail.nih.gov.