Mechanisms of Disease in Preterm Labor and Complications of Prematurity; Prenatal Diagnosis of Congenital Anomalies

Roberto Romero, MD, Head, Program in Perinatal Research and Obstetrics
Samuel S. Edwin, PhD, Senior Research Assistant
Francesca Gotsch, MD, Postdoctoral Fellow

The current taxonomy of disease in obstetrics is largely based on the clinical presentation of the mother rather than on the mechanism of disease responsible for clinical manifestations. Thus, the term “preterm labor” does not convey whether the condition is caused by infection, vascular insult, uterine overdistension, stress, or some other pathologic process. The same applies to other pregnancy complications such as preeclampsia, small for gestational age, and fetal death. We proposed that obstetrical disorders responsible for maternal death and perinatal morbidity and mortality are syndromes (hence, the designation the Great Obstetrical Syndromes) whose key features are (1) multiple etiology, (2) long preclinical stage, (3) frequent fetal involvement, (4) clinical manifestation that is often adaptive in nature, and (5) predisposition to a particular syndrome influenced by gene-environment interaction. Congenital anomalies continue to be a leading cause of perinatal mortality in the United States, many of which we can now detect prenatally with ultrasound. We use three- and four-dimensional ultrasound and develop novel approaches and algorithms aimed at improving the prenatal diagnosis of congenital anomalies, particularly congenital heart disease. We also combine sonographic and biochemical parameters to identify patients at increased risk of preeclampsia.

Proteomic analysis of amniotic fluid to identify women with preterm labor and intraamniotic inflammation/infection: novel computational method to analyze mass-spectrometric profiling

Romero, Gotsch, Edwin; in collaboration with Espinoza, Rogers, Moser, Nien, Kusanovic, Erez, Gomez, Hassan

High-dimensional biology techniques are expected to improve our understanding of the physiology or mechanisms of disease of various biological processes and to help develop new diagnostic, prognostic, and therapeutic tools in medicine. These new techniques include genomics, transcriptomics, proteomics, and metabolomics, which allow simultaneous examination of changes in the genome (DNA), transcriptome (mRNA), proteome (proteins), or metabolome (metabolites). Examination of the amniotic fluid proteome has been used to identify biomarkers for intra-amniotic inflammation and those that may be useful in predicting the outcome of patients with spontaneous preterm labor. In a cross-sectional study, we combined a novel computational method of pattern discovery with mass-spectrometric proteomic profiling of amniotic fluid to identify biomarkers of intra-amniotic infection/inflammation (IAI) in patients with spontaneous preterm labor and intact membranes who delivered at term and patients who delivered preterm with IAI. We conducted the proteomic profiling through several simultaneous surface-enhanced laser desorption/ionization (SELDI) mass-spectrometry conditions, which we combined into a single proteomic fingerprint. We used a novel computational approach to reduce the complexity of the data and identify individual features as well as patterns related to the clinical phenotype. At an overall accuracy of 90 percent (28/31), 39 unique mass-spectrometric peaks discriminated between patients with preterm labor/delivery with IAI and patients with preterm labor and term delivery. We have thus demonstrated that proteomic analysis of amniotic fluid allowed the identification of mass-spectrometry features that can distinguish patients with preterm labor with IAI from patients with preterm labor without inflammation or infection who subsequently delivered at term.

Romero R, Espinoza J, Rogers WT, Moser A, Nien JK, Kusanovic JP, Gotsch F, Erez O, Gomez R, Edwin S, Hassan SS. Proteomic analysis of amniotic fluid to identify women with preterm labor and intra-amniotic inflammation/infection: the use of a novel computational method to analyze mass spectrometric profiling. J Matern Fetal Neonatal Med 2008;21:367-388.

Detection of a microbial biofilm in intra-amniotic infection

Romero, Gotsch; in collaboration with Kusanovic, Erez, Kim CJ, Espinoza, Gonçalves, Vaisbuch, Mazaki-Tovi, Hassan, Costerton

We previously described the sonographic finding of dense aggregates of particulate matter in the amniotic fluid close to the uterine cervix of (1) women with an episode of preterm labor and (2) asymptomatic patients at risk for spontaneous preterm delivery. We proposed the term amniotic fluid “sludge” to refer to this sonographic finding and provided evidence that sludge is an independent risk factor for impending and early preterm delivery, preterm prelabor rupture of membranes (PROM), histologic chorioamnionitis, and microbial invasion of the amniotic cavity. Until now, intra-amniotic infection has been attributed to planktonic (free-floating) bacteria. The biofilm theory, however, states that most bacteria growing in exopolymeric matrix–enclosed biofilms differ from their planktonic counterparts. Microbial biofilms, which are communities of sessile microorganisms formed by cells irreversibly attached to a substratum or interface or to each other and embedded in a hydrated matrix of extracellular polymeric substances, have been implicated in over 80 percent of human infections, such as periodontitis, urethritis, endocarditis, and device-associated infections. To determine the nature of amniotic fluid sludge, we have been collecting the material by transvaginal needle amniotomy under ultrasound guidance in patients with imminent preterm delivery. Amniotic fluid sludge is macroscopically similar to pus and is associated with a markedly elevated white blood cell count in amniotic fluid as well as with positive Gram stain and a culture positive for microorganisms. In a case of spontaneous preterm labor and clinical chorioamnionitis at 28 weeks’ gestation, we retrieved amniotic fluid sludge and identified bacteria with scanning electron microscopy and fluorescence in situ hybridization for conserved regions of the microbial genome; we identified the exopolymeric matrix histochemically with the wheat germ agglutinin lectin method. We imaged amniotic fluid sludge with confocal laser scanning microscopy and scanning electron microscopy, which allowed the identification of bacteria embedded in an amorphous material and inflammatory cells. Our work represents the first report of a microbial biofilm in the amniotic cavity. The recognition that bacteria can form biofilms within the amniotic cavity is important because the diagnosis of microbial invasion in the presence of biofilms is extremely difficult, and current cultivation techniques to detect such infections are limited. Thus, the frequency of intra-amniotic infection may be underestimated, and molecular microbiologic techniques will be necessary to improve diagnosis. Moreover, bacteria within biofilms exhibit increased resistance to antimicrobial agents. Thus, the difficulties in the treatment of intra-amniotic infection may be attributable to the refractoriness of biofilms to conventional antibiotic treatment.

Romero R, Schaudinn C, Kusanovic JP, Gorur A, Gotsch F, Webster P, Nhan-Chang CL, Erez O, Kim CJ, Espinoza J, Gonçalves LF, Vaisbuch E, Mazaki-Tovi S, Hassan SS , Costerton JW. Detection of a microbial biofilm in intraamniotic infection. Am J Obstet Gynecol 2008;198:1335.e1-e5.

Soluble receptor for advanced glycation end products and endogenous secretory RAGE in amniotic fluid: modulation by infection and inflammation

Romero, Gotsch, Edwin; in collaboration with Espinoza, Hassan, Kusanovic, Erez

Advanced glycation end products (AGE) have been implicated in the pathogenesis of diabetes, renal failure, and aging. The receptor for advanced glycation end products (RAGE) has been proposed to participate in innate and adaptive immune responses. RAGE can induce the production of proinflammatory cytokines and chemokines as well as neutrophil chemotaxis in a manner that may be suppressed or stimulated by the soluble form of RAGE (sRAGE) and endogenous secretory RAGE (esRAGE). RAGE is expressed in the amnion epithelium, extravillous trophoblast, and decidual cells in patients without chorioamnionitis as well as in the neutrophils in the choriodecidua in cases of histologic chorioamnionitis. Our group conducted a study to determine if intra-amniotic infection/inflammation is associated with changes in the amniotic fluid concentration of sRAGE and esRAGE in the mid-trimester of pregnancy, in term and preterm labor, and in patients with preterm PROM. The amniotic fluid concentrations of sRAGE and esRAGE increased as a function of gestational age and decreased in the presence of labor at term; they were significantly elevated in the presence of intraamniotic infection/inflammation in patients with preterm labor and intact membranes and patients with preterm PROM, suggesting that changes in the amniotic fluid concentration of sRAGE and esRAGE may represent part of the immune response to intra-amniotic infection/inflammation.

Romero R, Espinoza J, Hassan S, Gotsch F, Kusanovic JP, Avila C, Erez O, Edwin S, Schmidt AM. Soluble receptor for advanced glycation end products (sRAGE) and endogenous secretory RAGE (esRAGE) in amniotic fluid: modulation by infection and inflammation. J Perinat Med 2008;36:388-398.

The anti-inflammatory limb of the immune response in preterm labor, intra-amniotic infection/inflammation, and spontaneous parturition at term: a role for interleukin-10 (IL-10)

Romero, Gotsch, Edwin; in collaboration with Kusanovic, Erez, Espinoza, Kim CJ, Vaisbuch, Than, Mazaki-Tovi, Gomez, Mittal, Hassan

Spontaneous parturition at both term and preterm labor are characterized by inflammation in the cervix, membranes, and myometrium. The anti-inflammatory limb of the immune response is crucial for dampening inflammation. In particular, the anti-inflammatory cytokine IL-10 participates in negative feedback to reduce inflammation. We designed a cross-sectional study of 301 pregnant women to determine whether there is a relationship between amniotic fluid concentrations of IL-10 and gestational age, parturition (term and preterm), and intra-amniotic infection/inflammation. We detected IL-10 in amniotic fluid and noted that its concentration did not change with gestational age from mid-trimester to term. Labor at term, however, was associated with a significantly elevated amniotic fluid IL-10 concentration. Moreover, the presence of intra-amniotic infection/inflammation was associated with a significantly higher amniotic fluid concentration of IL-10 in patients at term and patients with preterm labor with intact membranes as compared with patients without intra-amniotic infection/inflammation. The results suggest that IL-10 plays an important role in the control of labor and regulation of the immune response in vivo by initiating actions that dampen inflammation.

Gotsch F, Romero R, Kusanovic JP, Erez O, Espinoza J, Kim CJ, Vaisbuch E, Than NG, Mazaki-Tovi S, Chaiworapongsa T, Mazor M, Yoon BH, Edwin S, Gomez R, Mittal P, Hassan SS, Sharma S. The anti-inflammatory limb of the immune response in preterm labor, intra-amniotic infection/inflammation, and spontaneous parturition at term: a role for interleukin-10. J Matern Fetal Neonatal Med 2008;21:529-547.

Visfatin/Pre-B cell colony-enhancing factor in amniotic fluid in normal pregnancy, spontaneous labor at term, preterm labor, and prelabor rupture of membranes: an association with subclinical intrauterine infection in preterm parturition

Romero, Gotsch, Edwin; in collaboration with Mazaki-Tovi, Kusanovic, Erez, Mittal, Than, Vaisbuch, Chaiworapongsa, Nien, Gomez, Espinoza

Compelling evidence supports the view that cytokines play a central role in the mechanism of infection-induced preterm labor. Visfatin is a novel adipokine produced by visceral adipose tissue. The expression of its gene is elevated in several inflammatory conditions, including sepsis, metabolic syndrome, and rheumatoid arthritis. Visfatin is expressed by amniotic epithelium, cytotrophoblast, and decidua and overexpressed in fetal membranes exposed to mechanical stress and/or proinflammatory stimuli; its expression by fetal membranes is dramatically upregulated after normal spontaneous labor. We conducted a study to determine whether visfatin is detectable in amniotic fluid and whether its concentration changes with gestational age, spontaneous labor, and preterm PROM and in the presence of microbial invasion of the amniotic cavity. We found that visfatin is a physiologic constituent of amniotic fluid and that its concentrations in amniotic fluid increase with advancing gestational age and in patients with microbial invasion of the amniotic cavity, regardless of membrane status. This is the first in vivo study describing the presence of visfatin in amniotic fluid and its association with acute infection in humans. The findings suggest that visfatin plays a role in normal gestation as well as in the inflammatory response associated with infection. Increased visfatin secretion from fetal membranes, and possibly from intra-amniotic leukocytes in the presence of infection, may represent an attempt to protect the epithelial cells from apoptosis.

Mazaki-Tovi S, Romero R, Kusanovic JP, Erez O, Gotsch F, Mittal P, Than NG, Nhan-Chang CL, Hamill N, Vaisbuch E, Chaiworapongsa T, Edwin SS, Nien JK, Gomez R, Espinoza J, Kendal-Wright C, Hassan SS, Bryant-Greenwood G. Visfatin/Pre-B cell colony-enhancing factor in amniotic fluid in normal pregnancy, spontaneous labor at term, preterm labor and prelabor rupture of membranes: an association with subclinical intrauterine infection in preterm parturition. J Perinat Med 2008;36:485-496.

Region-specific gene expression profiling: novel evidence for biological heterogeneity of the human amnion

Romero, Gotsch; in collaboration with Han, Kim JS, Tarca, Kusanovic, Mittal, Hassan, Kim CJ

The amnion plays an important role during pregnancy and parturition. Though referred to as a single structure, this fetal tissue is regionally divided into placental amnion, reflected amnion, and umbilical amnion. Our empirical observations regarding the regional histological differences in meconium-exposed placentas, especially between placental amnion and reflected amnion, led us to hypothesize that the human amnion is biologically heterogeneous depending on its anatomical region in the placenta. We compared the gene expression profiles of placental amnion and reflected amnion in patients at term with and without spontaneous labor. Real-time quantitative reverse transcriptase (RT) PCR revealed a higher expression of IL-1beta mRNA in reflected amnion than in placental amnion in patients at term not in labor but not in patients in labor. Extended screening with microarray showed differential expression of 17 genes in labor. Interestingly, 839 genes were differentially expressed between placental and reflected amnion. Pathway analysis identified 19 signaling pathways, such as mitogen-activated protein kinase and transforming growth factor–beta pathways associated with region. Lipopolysaccharide (LPS ) treatment of the amnion explants showed more robust activation of mitogen-activated protein kinase 3/1 (extracellular signal-regulated kinase 1/2) in placental amnion of patients at term not in labor but not in patients in labor. Placental amnion from term–no labor and term–in labor cases showed a significant difference in the amplitude of IL-1beta mRNA induction by LPS. We report that the anatomical region of the amnion has a substantial impact on its transcriptional program and biological properties and that a particular feature of placental amnion is labor-associated switching to a pro-inflammatory signature. Our study’s novel observations strongly suggest that the apparently homogeneous amnion is biologically heterogeneous and compartmentalized, with implications for the physiology of pregnancy and parturition.

Han YM, Romero R, Kim JS, Tarca AL, Kim SK, Draghici S, Kusanovic JP, Gotsch F, Mittal P, Hassan S, Kim CJ. Region-specific gene expression profiling: novel evidence for biological heterogeneity of the human amnion. Biol Reprod 2008;79:954-961.

Gene expression profiling demonstrates a novel role for fetal fibrocytes and the umbilical vessels in human feto-placental development

Romero, Gotsch; in collaboration with Kim JS, Tarca, Han, Mittal, Kusanovic, Hassan, Kim CJ

The fetal inflammatory response syndrome (FIRS) is characterized by elevated IL-6 concentrations in the fetal plasma and multi-organ involvement. It is associated with an increased risk of preterm delivery and both short- and long-term perinatal morbidities, such as chronic lung disease and cerebral palsy. Acute funisitis, a component of acute chorioamnionitis, is the histologic hallmark of FIRS. A key feature of funisitis is acute umbilical vasculitis. Interestingly, umbilical phlebitis almost invariably precedes umbilical arteritis. The mechanisms responsible for the higher inflammatory response in the umbilical vein (UV) versus the umbilical arteries (UA) remain to be elucidated. Accordingly, we hypothesized that there is an intrinsic difference in the pro-inflammatory response between UA and UV. We collected and examined segments of the UA and UV from women with normal pregnancies at term not in labor, women with normal pregnancies at term in labor, women with preterm labor/delivery, women with intrauterine growth restriction (IUG R) at term, and women with preterm IUG R. Real-time quantitative RT-PC R and microarray analysis revealed higher expression of IL-1β and IL-8 mRNA in the UV than in the UA and differential expression of 567 genes between the UA and UV associated with distinct biological processes, including the immune response. Unexpectedly, HLA-DR(+) cells migrated via the umbilical vessels into Wharton’s jelly more frequently in the UV than in the UA. A significant proportion of these cells co-expressed CD45 and type I pro-collagen and acquired CD163 or α-smooth muscle actin immunoreactivity in Wharton’s jelly. We also found migrating cells in the chorionic and stem villous vessels. Furthermore, the extent of migration increased with advancing gestation but diminished in cases of IUGR. Our observations strongly suggest that circulating fetal fibrocytes, routing via umbilical and placental vessels, are a reservoir for key cellular subsets in the placenta. For the first time, we report fibrocytes in the human umbilical cord and placenta as well as a novel role for both circulating fetal cells and the umbilical vessels in placental development, which is disturbed in IUGR.

Kim JS, Romero R, Tarca AL, Lajeunesse C, Han YM, Kim MJ, Suh YL, Draghici S, Mittal P, Gotsch F, Kusanovic JP, Hassan S, Kim CJ. Gene expression profiling demonstrates a novel role for fetal fibrocytes and the umbilical vessels in human fetoplacental development. J Cell Mol Med 2008;12:1317-1330.

Galectin-1 expression in preterm PROM, histologic chorioamnionitis, severe preeclampsia, and small-for-gestational age neonates

Romero, Gotsch, Edwin; in collaboration with Than, Han, Tarca, Erez, Kusanovic, Montenegro, Mazaki-Tovi, Espinoza, Hassan

Galectin-1 is an anti-inflammatory protein with pleiotropic intra- and extracellular functions; it is expressed by chorioamniotic membranes and the decidua and overexpressed in activated immune and endothelial cells as well as in sites of inflammation, such as fibroblasts in chronic pancreatitis. Galectin-1 can also facilitate immune tolerance and tumor immune escape and has been successfully used for the suppression of experimental autoimmune diseases as well as for graft-versus-host disease in murine models. Given that an abnormal immune response in some pregnancy complications might be associated with changes in placental and membrane expression of galectin-1, we designed a series of studies to determine galectin-1 mRNA and protein expression in (1) the chorioamniotic membranes in women with a normal pregnancy and women with preterm PROM with and without histologic chorioamnionitis and (2) the placenta of women with normal pregnancies, women with severe preeclampsia, and women who delivered a small-for-gestational age (SGA) neonate. In normal term placentas, we detected galectin-1 mRNA or immunofluorescence signals in the trophoblasts, villous stromal cells, Hofbauer cells, endothelial cells of the villous blood vessels, and the villous stroma. In patients with preterm PROM, we detected galectin-1 mRNA and protein in the amniotic epithelium, chorioamniotic fibroblasts/myofibroblasts and macrophages, chorionic trophoblasts, and decidual stromal cells. Patients with preterm PROM and histologic chorioamnionitis exhibited 2-fold–higher galectin-1 mRNA expression in the fetal membranes than patients with preterm PROM without histologic chorioamnionitis. Moreover, histologic chorioamnionitis was associated with strong galectin-1 immunostaining in amniotic epithelium, chorioamniotic mesodermal cells, and apoptotic bodies. These findings suggest that galectin-1 may be involved in regulating inflammatory responses to chorioamniotic infection. Furthermore, placental galectin-1 mRNA expression was significantly higher in severe preeclampsia (with or without SGA) than in controls and SGA; trophoblasts in placentas of patients with severe preeclampsia exhibited the most intense galectin-1 immunostaining. Placental galectin-1 expression is higher in severe preeclampsia than in normal pregnancy regardless of the presence of SGA but is not altered in SGA without preeclampsia. This is the first report of placental expression and localization of galectin-1 mRNA. We propose that the increased placental expression of galectin-1 in patients with severe preeclampsia may represent a fetal response to an exaggerated systemic maternal inflammation. Thus, galectin-1 may be implicated in maternal-fetal immune tolerance.

Than NG, Erez O, Wildman DE, Tarca AL, Edwin SS, Abbas A, Hotra J, Kusanovic JP, Gotsch F, Hassan SS, Espinoza J, Papp Z, Romero R. Severe preeclampsia is characterized by increased placental expression of galectin-1. J Matern Fetal Neonatal Med 2008;21:429-442.
Than NG, Kim SS, Abbas A, Han YM, Hotra J, Tarca AL, Erez O, Wildman DE, Kusanovic JP, Pineles B, Montenegro D, Edwin SS, Mazaki-Tovi S, Gotsch F, Espinoza J, Hassan SS, Papp Z, Romero R. Chorioamnionitis and increased galectin-1 expression in PPROM—an anti-inflammatory response in the fetal membranes? Am J Reprod Immunol 2008;60:298-311.

First-trimester maternal serum PP13 in the risk assessment for preeclampsia

Romero, Gotsch, Edwin; in collaboration with Kusanovic, Than, Erez, Espinoza, Gomez, Nien, Hassan

Placental Protein 13 (PP 13) is a member of the galectin family and is predominantly expressed by the placenta, specifically by the syncytiotrophoblast, where it is localized on the brush-border membrane at the maternal-fetal interface. Recently, we found that maternal serum PP 13 concentrations were significantly reduced during the first trimester among women who subsequently developed preeclampsia. We designed a nested case-control study to determine whether first-trimester maternal serum PP13 concentrations may be used to assess risk for preeclampsia. Our study included 50 patients with preeclampsia and 250 patients with normal pregnancies from whom we collected maternal blood samples between 8 and 13 weeks of gestation. The results showed that serum PP13 concentration in the first trimester was significantly lower in patients who developed preterm and early-onset preeclampsia than in those with normal pregnancies and that, when specificity was fixed at 80 percent, a cutoff of 0.39 MoM¹ had a sensitivity of 100 and 85 percent, respectively, for prediction of early-onset preeclampsia and preterm preeclampsia. We concluded that maternal serum concentration of PP13 in the first trimester appears to be a reasonable marker for risk assessment for preterm preeclampsia.

Romero R, Kusanovic JP, Than NG, Erez O, Gotsch F, Espinoza J, Edwin S, Chefetz I, Gomez R, Nien JK, Sammar M, Pineles B, Hassan SS , Meiri H, Tal Y, Kuhnreich I, Papp Z, Cuckle HS . First-trimester maternal serum PP13 in the risk assessment for preeclampsia. Am J Obstet Gynecol 2008;199:122.e1-e11.

A longitudinal study of angiogenic and anti-angiogenic factors in normal pregnancy and patients destined to develop preeclampsia and deliver an SGA neonate

Romero, Gotsch, Edwin; in collaboration with Erez, Espinoza, Kusanovic, Nien, Chaiworapongsa, Mittal, Mazaki-Tovi, Than, Gomez, Hassan

An imbalance between pro-angiogenic factors, i.e., vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), and anti-angiogenic factors, i.e., soluble VEGF receptor-1 (sVEG FR-1, also referred to as sFlt1) and soluble endoglin (s-Eng), is involved in the pathophysiology of preeclampsia. We designed a longitudinal nested case-control study to determine whether changes in maternal plasma concentration of these angiogenic and anti-angiogenic factors differ before the development of disease in patients with normal pregnancies and patients destined either to develop preeclampsia (preterm and term) or deliver an SGA neonate. The study included 144 singleton pregnancies from which we collected longitudinal blood samples at each prenatal visit, scheduled at 4-week intervals from the first or early second trimester until delivery. Patients destined to deliver an SGA neonate had higher plasma concentrations of s-Eng throughout gestation than those with normal pregnancies. Similarly, patients destined to develop preterm preeclampsia and term preeclampsia exhibited significantly higher concentrations of s-Eng than those with normal pregnancies at 23 and 30 weeks, respectively, and a significantly higher plasma concentration of sVEGFR-1 at 26 and 29 weeks of gestation, respectively. Moreover, patients destined to develop preeclampsia (term or preterm) and those who delivered an SGA neonate had lower plasma concentrations of PlGF than those with a normal pregnancy throughout gestation, and the maternal plasma concentration of this analyte was detectable later among patients with pregnancy complications than in normal pregnant women. Interestingly, we observed no significant differences in the plasma concentrations of sVEG FR-1 between patients destined to deliver an SGA neonate and those with normal pregnancies. Our longitudinal study demonstrated that changes in the maternal plasma concentration of s-Eng, sVEGFR-1, and PlGF precede the clinical presentation of preeclampsia but that only changes in s-Eng and PlGF precede the delivery of an SGA neonate. The differences in the profile of angiogenic and anti-angiogenic response to intrauterine insults may determine whether a patient will deliver an SGA neonate, develop preeclampsia, or both.

Given that changes in the maternal plasma concentration of angiogenic and anti-angiogenic factors precede the clinical presentation of preeclampsia and the delivery of an SGA neonate, we aimed to determine whether changes in the profile of angiogenic and anti-angiogenic factors in maternal plasma between the first and second trimesters are associated with a high risk for the subsequent development of preeclampsia and/or delivery of an SGA neonate. We conducted a case-control study that included 402 singleton pregnancies in the following groups: patients with normal pregnancies, patients who delivered an SGA neonate, and patients who developed preeclampsia. The study included two samples per patient, with the first sample obtained between 6 and 15 weeks of gestation (“first trimester” sample) and the second sample obtained between 20 and 25 weeks of gestation (“second trimester” sample). We compared changes in the maternal plasma concentrations of the angiogenic and anti-angiogenic factors among normal patients and those destined to develop preeclampsia or deliver an SGA neonate while adjusting for maternal age, nulliparity, and body mass index. We used general linear models and polytomous logistic regression models to relate the analyte concentrations, ratios, and products to the subsequent development of preeclampsia and SGA. Our study showed that an increase in the maternal plasma concentration of s-Eng between the first and second trimesters conferred risk for the development of preterm preeclampsia and SGA . Similarly, an increase in the maternal plasma concentration of sVEG FR-1 between the first and second trimester conferred risk for the development of preterm preeclampsia, whereas a subnormal increase in maternal plasma PlGF concentration between the first and second trimester was a risk factor for the subsequent development of preterm and term preeclampsia. In addition, the combination of the three analytes into a pro-angiogenic versus anti-angiogenic ratio [PlGF/(s-Eng x VEGFR-1)] conferred an elevated risk for subsequent development of preterm preeclampsia. In conclusion, changes in the maternal plasma concentrations of s-Eng, sVEGFR-1, PlGF, or their ratios between the first and second trimester confer an increased risk to deliver an SGA neonate and/or develop preeclampsia.

Erez O, Romero R, Espinoza J, Fu W, Todem D, Kusanovic JP, Gotsch F, Edwin S, Nien JK, Chaiworapongsa T, Mittal P, Mazaki-Tovi S, Than NG, Gomez R, Hassan SS. The change in concentrations of angiogenic and anti-angiogenic factors in maternal plasma between the first and second trimesters in risk assessment for the subsequent development of preeclampsia and small-for-gestational age. J Matern Fetal Neonatal Med 2008;21:279-287.
Romero R, Nien JK, Espinoza J, Todem D, Fu W, Chung H, Kusanovic JP, Gotsch F, Erez O, Mazaki-Tovi S, Gomez R, Edwin S, Chaiworapongsa T, Levine RJ, Karumanchi SA. A longitudinal study of angiogenic (placental growth factor) and anti-angiogenic (soluble endoglin and soluble vascular endothelial growth factor receptor-1) factors in normal pregnancy and patients destined to develop preeclampsia and deliver a small for gestational age neonate. J Matern Fetal Neonatal Med 2008;21:9-23.

Maternal-plasma sVEGFR-1 concentration is elevated in SGA, and the magnitude of the increase relates to Doppler abnormalities in the maternal and fetal circulation

Romero, Gotsch, Edwin; in collaboration with Chaiworapongsa, Espinoza, Gonçalves, Kusanovic, Erez, Than, Hassan

Preeclampsia and pregnancy complicated by SGA fetuses share some pathophysiologic derangements, including failure of physiologic transformation of the spiral arteries, endothelial cell dysfunction, and leukocyte activation. An antagonist to vascular endothelial growth factor and placental growth factor, sVEGFR-1, has been implicated in the pathophysiology of preeclampsia (see above). We designed a study with two purposes. The first was to determine whether plasma concentrations of sVEGFR-1 in mothers with SGA fetuses without preeclampsia at the time of diagnosis differ from plasma concentrations of sVEGFR-1 in patients with preeclampsia or in patients with a normal pregnancy. The second purpose was to examine the relationship between plasma concentrations of sVEGFR-1 and Doppler velocimetry of uterine and umbilical arteries in patients with preeclampsia and patients with SGA. Mothers with SGA fetuses had a higher mean plasma concentration of sVEG FR-1 than normal pregnant women but a lower concentration than patients with preeclampsia. Among mothers with preeclampsia and those with SGA fetuses in whom Doppler velocimetry was performed, those with abnormalities in both uterine and umbilical artery velocimetry had the highest mean deviation of sVEGFR-1 plasma concentrations from the mean for gestational age. These observations provide support for the participation of the soluble receptor of VEGF in the pathophysiology of SGA with abnormal uterine artery Doppler velocimetry and preeclampsia.

Chaiworapongsa T, Espinoza J, Gotsch F, Kim YM, Kim GJ, Gonçalves LF, Edwin S, Kusanovic JP, Erez O, Than NG, Hassan SS, Romero R. The maternal plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated in SGA and the magnitude of the increase relates to Doppler abnormalities in the maternal and fetal circulation. J Matern Fetal Neonatal Med 2008;21:25-40.

Distinct genomic signatures of adaptation in pre- and postnatal environments during human evolution

Romero; in collaboration with Erez, Goodman, Grossman

The human genome evolution project seeks to reveal the genetic underpinnings of key phenotypic features that are distinctive of humans, such as a greatly enlarged cerebral cortex, slow development, and long life spans. We hypothesized that adaptive genotypic changes during earlier periods of evolutionary history also helped shape the distinctive human phenotype. Focusing predominantly on genotypic changes during the 6-million-year descent from the last common ancestor of humans and chimpanzees, we set out to identify the positively selected genetic changes that may have helped shape key, distinctive (and, in some cases, unique) phenotypic features of humans. Using comparative genome sequence data from 10 vertebrate species, we found a signature of human ancestry–specific adaptive evolution in 1,240 genes during their descent from the last common ancestor with rodents. We also found that the signature of adaptive evolution differs significantly for highly expressed genes in human fetal and adult-stage tissues. Functional annotation clustering showed that, on the ape stem lineage, an especially evident, adaptively evolved biological pathway contains genes that function in mitochondria, that are crucially involved in aerobic energy production, and that are highly expressed in two energy-demanding tissues—heart and brain. In addition, the ape stem lineage evidenced adaptive evolution among genes associated with human autoimmune and agingrelated diseases. Interestingly, during more recent human descent, the adaptively evolving, highly expressed genes in the fetal brain are involved in mediating neuronal connectivity. Our comparison of adaptively evolving genes from pre- and postnatal-stage tissues suggests that different selective pressures act on the development as opposed to the maintenance of the human phenotype.

Uddin M, Goodman M, Erez O, Romero R, Liu G, Islam M, Opazo JC, Sherwood CC, Grossman LI, Wildman DE. Distinct genomic signatures of adaptation in pre- and postnatal environments during human evolution. Proc Natl Acad Sci USA 2008;105:3215-3220.

The human progesterone receptor shows evidence of adaptive evolution associated with its ability to act as a transcription factor

Romero; in collaboration with Erez, Goodman, Grossman

The steroid hormone progesterone is a vital regulator of reproduction in mammals. In primates, progesterone participates in the regulation of normal menstrual cycles, ovulation, and embryo implantation. Many of progesterone’s biological actions are mediated through the progesterone receptor. The gene encoding the receptor (PGR) arose early in vertebrate evolution via a series of duplications of an ancestral estrogen receptor; it acts as a transcription factor and participates in the regulation of reproductive processes, including menstruation, implantation, pregnancy maintenance, parturition, mammary development, and lactation. Unlike other mammals, primates do not exhibit progesterone withdrawal at the time of parturition. Given that progesterone-mediated reproductive features vary among mammals, PGR is an attractive candidate gene for studies of adaptive evolution. We sequenced the progesterone receptor–coding regions in a diverse range of species, including apes, Old World monkeys, New World monkeys, prosimian primates, and other mammals. We investigated three aspects of PGR evolution: (1) the strength of positive selection in human and chimpanzee lineages, (2) the prevalence of positive selection within primates, and (3) the specific isoforms and domains at which amino acids were replaced. Adaptive evolution occurred in the human and chimpanzee lineages, as evidenced by statistically significant higher non-synonymous substitution rates than synonymous substitution rates. We rarely observed the positive selection seen in other lineages. In humans, amino acid replacements occurred mostly in a region of the gene shown to inhibit the ability of the progesterone receptor to act as a transcription factor. Moreover, many of the non-synonymous substitutions in primates occurred in the N-terminus, suggesting that co-factor interaction surfaces might have been altered as reflected in altered progesterone-regulated gene transcriptional effects. It is possible that changes in the inhibitory function region uniquely modulate expression and interaction among the different progesterone isoforms in functional progesterone withdrawal during human and chimpanzee parturition. Positive selection provides the basis for the hypothesis that changes in structure and function of the progesterone receptor during evolution contribute to the diversity of primate reproductive biology, especially in parturition.

Chen C, Opazo JC, Erez O, Uddin M, Santolaya-Forgas J, Goodman M, Grossman LI, Romero R, Wildman DE. The human progesterone receptor shows evidence of adaptive evolution associated with its ability to act as a transcription factor. Mol Phylogenet Evol 2008;47:637-649.

Twin-to-twin transfusion syndrome: an anti-angiogenic state?

Romero, Gotsch, Edwin; in collaboration with Kusanovic, Espinoza, Nien, Kim CJ, Mittal, Erez, Mazaki-Tovi, Than, Gomez, Hassan

Twin-to-twin transfusion syndrome (TTS) affects approximately 10 to 15 percent of monochorionicdiamniotic twin pregnancies; its diagnosis is based on strict sonographic criteria. An imbalanced chronic blood flow between the donor and recipient twin through placental vascular anastomoses is the accepted pathophysiology of TTS. Recently, others demonstrated that VEGFR-1 mRNA is overexpressed in some cases of TTS only in the donor twin’s syncytiotrophoblast. We conducted a case control study to determine maternal plasma concentrations of the angiogenic factor PlGF and of the anti-angiogenic factors sVEGFR-1 and s-Eng in monochorionic-diamniotic pregnancies between 16 and 26 weeks’ gestation with and without TTS. As compared with patients without TTS, patients with TTS had significantly higher median plasma concentrations of s-Eng and sVEGFR-1 and lower median plasma concentrations of PlGF. This novel finding suggests that an anti-angiogenic state may be present in some cases of TTS.

Kusanovic JP, Romero R, Espinoza J, Nien JK, Kim CJ, Mittal P, Edwin S, Erez O, Gotsch F, Mazaki-Tovi S, Than NG, Soto E, Camacho N, Gomez R, Quintero R, Hassan SS. Twin-to-twin transfusion syndrome: an antiangiogenic state? Am J Obstet Gynecol 2008;198:382.e1-e8.

Individualized growth assessment of fetal thigh circumference using three-dimensional ultrasonography

Romero; in collaboration with Lee, Espinoza, Gonçalves

Estimated fetal weight and growth are usually determined by using formulas that incorporate fetal biometric parameters measured by two-dimensional ultrasound. Using three-dimensional ultrasonography, we conducted a prospective, longitudinal sonographic study of 30 fetuses beginning at 18 weeks’ menstrual age to develop individualized growth assessment (IGA) standards for upper and middle fetal thigh circumferences. We measured second-trimester sonographic parameters from three-dimensional volume data to establish IGA standards, subsequently predicted third-trimester growth trajectories and birth measurements for upper and middle fetal thigh circumferences, and determined the 95 percent ranges for Growth Potential Realization Index (GPRI) values for both types of thigh circumference. The 30 newborns showed no postnatal evidence of abnormal growth. Our measurements demonstrated that fetal thigh circumference may be evaluated and measured reliably with standard IGA methods. Both upper and middle thigh circumferences yielded similar results in the third trimester. However, the use of middle thigh circumferences improved neonatal thigh circumference predictions. Corresponding GPRI values for fetal middle thigh circumference are closer to the ideal value of 100 percent and may be used in calculations for assessing neonatal growth outcome.

Lee W, Deter RL, Sameera S, Espinoza J, Gonçalves LF, Romero R. Individualized growth assessment of fetal thigh circumference using three-dimensional ultrasonography. Ultrasound Obstet Gynecol 2008;31:520-528.

A systematic approach to the use of the multiplanar display in evaluation of abnormal vascular connections to the fetal heart using four-dimensional ultrasonography

Romero, Gotsch; in collaboration with Espinoza, Hassan, Kusanovic, Lee, Erez, Gonçalves

Congenital heart disease is the leading cause of infant death among newborns with congenital anomalies. Conotruncal anomalies represent one-fifth of all congenital heart defects diagnosed prenatally. Simultaneous display of standard views used in fetal echocardiography and their orthogonal planes may provide novel ultrasonographic views for examining the fetal heart and its vascular connections. We designed a study to determine the clinical utility of the multiplanar display in the examination of abnormal vascular connections to the fetal heart. We reviewed fourdimensional volume data sets, acquired with the spatiotemporal image correlation technique, from four patients with abnormal vascular connections to the fetal heart. We used multiplanar views of the fetal heart to display simultaneously the standard planes used in fetal echocardiography and their corresponding orthogonal planes. This method facilitated the identification of the abnormal vessels and their spatial relationships with other vascular structures. Thus, we concluded that multiplanar imaging may be used to assess abnormal vascular connections to the fetal heart and may provide novel ultrasonographic planes for fetal echocardiography with three- and four-dimensional ultrasonography.

Espinoza J, Hassan SS, Gotsch F, Kusanovic JP, Lee W, Erez O, Gonçalves LF, Schoen ML, Romero R. A systematic approach to the use of the multiplanar display in evaluation of abnormal vascular connections to the fetal heart using 4-dimensional ultrasonography. J Ultrasound Med 2007;26:1461-1467.

The use of inversion mode and three-dimensional manual segmentation in volume measurement of fetal fluid-filled structures: comparison with Virtual Organ Computeraided AnaLysis (VOCAL)

Romero; in collaboration with Kusanovic, Nien, Gonçalves, Espinoza, Lee, Erez

Volume measurements obtained by three-dimensional ultrasonography are considered more accurate than those obtained by two-dimensional ultrasonography that uses the ellipsoid formula, among other formulas. We conducted a study to compare agreement among three techniques (VOCAL, inversion mode, and manual segmentation) as well as inter- and intra-observer agreements for volume measurements of fetal fluid-filled structures. We obtained 50 three-dimensional volume data sets of the fetal stomach and bladder; two observers independently performed volume measurements. The three techniques yielded a high degree of reliability and good agreement. We obtained manual segmentation and inversion mode measurements significantly faster with the VOCAL technique than with the other techniques. We concluded that the inversion mode is a reliable method for volume calculations of fluid-filled organs, whereas manual segmentation may be used when volume measurements by VOCAL or the inversion mode are technically difficult to obtain.

Kusanovic JP, Nien JK, Gonçalves LF, Espinoza J, Lee W, Balasubramaniam M, Soto E, Erez O, Romero R. The use of inversion mode and 3D manual segmentation in volume measurement of fetal fluid-filled structures: comparison with Virtual Organ Computer-aided AnaLysis (VOCAL). Ultrasound Obstet Gynecol 2008;31:177-186.

¹Multiples of the median

Collaborators

Tinnakorn Chaiworapongsa, MD, Wayne State University School of Medicine, Detroit, MI
John W. Costerton, PhD, School of Dentistry, University of Southern California, Los Angeles, CA
Russell Deter, MD, Baylor College of Medicine, Waco, TX
Daniel B. DiGiulio, MD, Stanford University School of Medicine, Palo Alto, CA
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. Gonçalves, MD, Wayne State University School of Medicine, Detroit, MI
Morris Goodman, PhD, Wayne State University School of Medicine, Detroit, MI
Lawrence Grossman, PhD, Wayne State University School of Medicine, Detroit, MI
Yu Mi Han, 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
Juan Pedro Kusanovic, MD, Wayne State University School of Medicine, Detroit, MI
Wesley Lee, MD, William Beaumont Hospital, Royal Oak, MI
Shali Mazaki-Tovi, MD, Wayne State University School of Medicine, Detroit, MI
Pooja Mittal, MD, Wayne State University School of Medicine, Detroit, MI
Daniel Montenegro, BS, Wayne State University School of Medicine, Detroit, MI
Allen Moser, PhD, Cira Discovery Sciences, Inc., Philadelphia, PA
Jyh Kae Nien, MD, Sotero del Rio Hospital, Puente Alto, Chile
David A. Relman, MD, Stanford University School of Medicine, Palo Alto, CA
Wade Rogers, PhD, University of Pennsylvania School of Medicine, Philadelphia, PA
Jerome F. Strauss, II, MD, PhD, Virginia Commonwealth University, Richmond, VA
Adi Tarca, PhD, Wayne State University School of Medicine, Detroit, MI
Nandor Gabor Than, MD, PhD, Wayne State University School of Medicine, Detroit, MI
Edi Vaisbuch, MD, Wayne State University School of Medicine, Detroit, MI

For further information, contact jpowers@med.wayne.edu.