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

Roberto Romero, MD
  • Roberto Romero, MD, Head, Program in Perinatal Research and Obstetrics

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. The research agenda of the Program in Perinatology Research and Obstetrics (PPRO) includes the use of three- and four-dimensional ultrasound and the development of novel approaches and algorithms aimed at facilitating the prenatal diagnosis of congenital defects, in particular, the prenatal identification of congenital heart disease. This year, we emphasized the study of fetal growth and the development of formulae to improve the estimation of fetal weight based on three-dimensional parameters.

Microbial prevalence, diversity, and abundance in amniotic fluid in the Great Obstetrical Syndromes: a molecular and culture-based investigation

Estimation of the prevalence of microbial invasion of the amniotic cavity (MIAC) has been based largely on cultivation techniques. The limitations of such methods are well-established (they require knowledge of the conditions to support growth of microorganisms in vitro). Molecular techniques provide a means to discover microorganisms that may not have been cultivated. We analyzed the amniotic fluid (AF) of 204 women with pPROM (preterm premature rupture of membranes), using both cultivation and molecular methods. The prevalence of MIAC was 34% by culture, 45% by PCR, and 50% by both methods combined. The number of bacterial species revealed by PCR was greater than that by culture (44 vs. 14 species), including as-yet uncultivated taxa. Some taxa detected by PCR had been previously identified in the gastrointestinal tract and the mouth. Bacterial rDNA abundance exhibited a dose relationship with gestational age at delivery, and a positive PCR was associated with a lower gestational age at delivery and higher rates of respiratory distress syndrome and necrotizing enterocolitis. The study indicates that MIAC in pPROM is more common than previously recognized and, in some cases, associated with uncultivated taxa.

Infection has also been implicated in the pathogenesis of preeclampsia and as a potential cause of fetal growth restriction; yet, the associations between MIAC and preeclampsia and between MIAC and small-for-gestational-age (SGA) fetuses have not been characterized. AF from 62 subjects with preeclampsia and 52 subjects with an SGA neonate, not in labor, was analyzed using both cultivation and molecular methods. The rate of MIAC in preeclampsia was 1.6% based on culture, 8% based on PCR, and 9.6% based on both methods. All AF samples of patients with an SGA neonate were negative for microorganisms based on cultivation techniques, whereas 6% were positive based on PCR. The studies demonstrate that the prevalence of MIAC in preeclampsia and in SGA is low, suggesting that intra-amniotic infection plays a limited role in their pathophysiology.

Genetic association studies of maternal and fetal candidate genes in preterm labor with intact membranes, pPROM, and SGA fetuses

A study of the role of genomics in preterm birth (PTB) aims to determine whether there is a genetic predisposition to spontaneous PTB and delivery. We conducted a series of studies to determine whether maternal/fetal single nucleotide polymorphisms (SNPs) in candidate genes are associated with spontaneous PTB/delivery, pPROM, and SGA. We conducted genetic association studies in 223 mothers with PTB/delivery and 179 of their fetuses, 225 mothers with pPROM and 155 of their fetuses, 530 mothers and 436 fetuses that were SGA, and 599 mothers with a normal pregnancy and 628 of their fetuses. We studied 190 candidate genes and 775 SNPs. Single locus/haplotype association analyses were performed. The strongest single locus associations with PTB/delivery were IL-6 receptor 1 (fetus) and TIMP-2 (mother), which remained significant after correction for multiple comparisons. Global haplotype analysis indicated an association between a fetal DNA variant in insulin-like growth factor F2 and maternal alpha 3 type IV collagen isoform 1. Similarly, a SNP in TIMP-2 in mothers was significantly associated with pPROM, an association that remained significant after correction for multiple comparisons. Haplotypes for alpha 3 type IV collagen isoform precursor in the mother were associated with pPROM; multilocus analysis identified a 3-locus model for pPROM, which included maternal SNPs in collagen type I alpha 2, defensin alpha 5 gene, and endothelin 1. Among patients with an SGA fetus, the most significant single-locus association in mothers was a SNP in TIMP-2, while in the fetus it was a SNP in fibronectin 1 isoform 3 preproprotein (FN1). Haplotype analyses resulted in associations in alpha 1 type I collagen preproprotein in mothers and FN1 in fetuses. Multi-locus analyses identified a two-SNP model, with maternal variants COL5A2 and PLAU predicting SGA outcome correctly 59% of the time. The studies support the hypothesis that DNA variants can partially explain the risk of PTB, pPROM and SGA.

The frequency, clinical significance, and pathological features of chronic chorioamnionitis: a lesion associated with spontaneous preterm birth

The current diagnostic paradigm in surgical pathology distinguishes acute and chronic chorioamnionitis (CA). CA has been described as a rare lesion of the extraplacental membranes and chorionic plate. In an attempt to elucidate the pathologic basis of late preterm birth in which acute CA is rare, we systematically examined the placentas of patients experiencing preterm labor (PTL) with intact membranes and patients experiencing pPROM. The frequency of chronic CA was 34% in PTL and 39% in pPROM, which is higher than that observed in normal term placentas (8%). The median gestational age of cases with preterm chronic CA was higher than those with acute CA, suggesting that acute and chronic CA represent distinct pathologic clusters of the preterm parturition syndrome. We also found that the mRNA expression of CXCL9, CXCL10, and CXCL11 (T-cell chemokines) was elevated in the membranes with chronic CA, and that the median AF CXCL10 concentration was significantly higher in patients with chronic CA than in those without this lesion in the PTL and pPROM groups. The absence of microorganisms identified either by culture or by molecular methods suggests that this lesion does not have a bacterial origin. Our observations suggest that chronic CA may account for the pathology of a large number of spontaneous preterm deliveries. The infiltration of lymphocytes into a semi-allograft suggests an immunological mechanism akin to that of transplantation rejection and graft-versus-host disease in the chorioamniotic membranes.

A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia

Changes in maternal plasma concentrations of angiogenic (PlGF and VEGF) and anti-angiogenic factors (sEng and sVEGFR-1) precede the clinical presentation of preeclampsia. We conducted a longitudinal cohort study including 1622 singleton pregnant women to examine whether maternal plasma PlGF, sEng, and sVEGFR-1 concentrations during early pregnancy and midtrimester can assist in the identification of patients destined to develop preeclampsia. Higher likelihood ratios were provided by ratios of midtrimester plasma concentrations of PlGF, sEng, and sVEGFR-1 than single analytes. Individual angiogenic and anti-angiogenic factors did not perform well in the identification of preeclampsia as a whole. In contrast, a combination of these analytes such as the PlGF/sEng ratio, its delta, and slope gave the best predictive values with a sensitivity of 100%, a specificity of 98-99%, and likelihood ratios for a positive test of 57.6, 55.6, and 89.6, respectively, for predicting early-onset preeclampsia (less than 34 weeks), rendering these analytes suitable for the risk assessment of preeclampsia.

Metabolomics in premature labor: a novel approach to identifying patients at risk for preterm delivery

Biomarkers for PTL and delivery can be discovered through the analysis of the metabolic network of cells (metabolome). Characterization of the global changes of the metabolome is now possible by using metabolomics. We conducted a study to determine if metabolomic profiling of the AF can identify women with spontaneous PTL at risk for preterm delivery, regardless of the presence or absence of intra-amniotic infection/inflammation (IAI). We conducted two retrospective cross-sectional studies that included pregnant women with PTL who delivered at term, PTL without IAI who delivered preterm, and PTL with IAI. AF metabolic profiling was performed by combining gas and liquid chromatography, and mass spectrometry. Compounds were identified by using authentic standards. In the exploratory study, metabolomic profiling of the AF was able to identify patients as belonging to the correct clinical group with an overall accuracy of 96.3%. In the validation study, metabolomic profiling was able to identify patients as belonging to the correct clinical group with an accuracy of 88.5%. The metabolites responsible for the classification of patients in different clinical groups were identified. A preliminary draft of the human AF metabolome was generated (e.g. xenobiotic compounds).

Sex differences in fetal growth responses to maternal height and weight

Gender differences in fetal growth have been reported, but it is unknown whether fetal growth rates, a reflection of genetic and environmental factors, express sexually dimorphic sensitivity to the mother. Our group investigated the homogeneity of male and female growth responses to maternal height and weight in 3,495 uncomplicated singleton pregnancies (1,814 males; 1,681 females) followed longitudinally. Gender modified the effects of maternal height and weight on fetal growth rates and birth weight. Among males, tallest maternal height influenced fetal weight growth before 18 weeks of gestation, and pre-pregnancy maternal weight and body mass index subsequently had influence, a finding that was not seen in females. Additionally, males were more sensitive to maternal weight among shorter mothers and more responsive to maternal height among lighter mothers, compared with females. A male advantage averaging 60g occurred among neonates of the shortest and lightest mothers, compared with 150g and 191g among short and heavy mothers, and tall and light-weight mothers, respectively. The study indicated that gender differences in response to maternal size are under-appreciated sources of variation in fetal growth studies and may reflect differential growth strategies.

Iliac crest angle: a novel sonographic parameter for the prediction of Down syndrome risk during the second trimester of pregnancy

We investigated a new sonographic technique for the display and measurement of the fetal iliac crest angle (ICA) and determined its value for estimating the risk of Down syndrome during the midtrimester. Three-dimensional ultrasonography (3DUS) of the fetal pelvis was performed during genetic amniocenteses. Two different ICAs were measured from a coronal projection of the fetal pelvis (ICA-coronal 1 and ICA-coronal 2). We also measured axial inner (ICA-inner), middle (ICA-middle) and outer (ICA-outer) ICAs. Ninety-four normal fetuses and 19 fetuses with Down syndrome were examined. The ICA-middle and ICA-coronal 2 parameters were the most reproducible measurements. The mean ICA-middle measurement for fetuses with Down syndrome was significantly greater than that for normal subjects (94.5 vs. 83.1 degrees). The mean ICA-coronal 2 angle measurement for fetuses with Down syndrome was slightly greater than that for normal subjects (57.9 vs. 51.9 degrees). A multiple logistic regression model including ICA-middle and ICA-coronal 2 provided a predictive ability of 88.1%. This combination had a sensitivity of 94.4% for a false-positive rate of 5% in the detection of Down syndrome. We propose that standardized ICA measurements of the fetal pelvis during the midtrimester of pregnancy can be used to identify some fetuses at risk for trisomy 21.

New fetal weight estimation models using fractional limb volume

Evaluation of the soft tissue of fetal limbs with 3DUS is feasible. The use of fractional limb volume addresses the technical limitations of fetal weight estimation. Our group conducted a study to determine the accuracy and precision of new fetal weight estimation models, based on fractional limb volume and conventional two-dimensional (2D) sonographic measurements during the second and third trimesters. A prospective cross-sectional study of 271 fetuses was performed using 3DUS and measuring biparietal diameter (BPD), abdominal circumference (AC) and femoral diaphysis length (FDL) plus fractional arm volume (AVol) and fractional thigh volume (TVol) within four days prior to delivery. Weighted multiple linear regression analysis was used to develop modified Hadlock models and new models using transformed predictors that included soft tissue parameters for estimating birth weight. Six new models were highly accurate, with mean percent differences not significantly different from zero. Model 3 (natural logarithm [ln] of BPD, AC and AVol) and Model 6 (ln of BPD, AC and TVol) provided the most precise weight estimations (random error=6.6% of actual birth weight) as compared with 8.5% for the best original Hadlock model and 7.6% for a modified Hadlock model. This study demonstrates that the precision of fetal weight estimation can be improved by adding fractional limb volume measurements to conventional 2D biometry. These new models may offer novel insight into the contribution of soft tissue development to weight estimation.

Non-invasive fetal lung assessment using diffusion-weighted imaging

The use of magnetic resonance imaging (MRI) during pregnancy is increasing. Diffusion-weighted imaging (DWI) provides an image contrast that is based on the molecular motion of water—a process that can be altered by the presence of disease. The apparent diffusion coefficient (ADC) is based on these principles and reflects water movement within the tissue environment. We conducted a study to develop a reproducible method for estimating the diffusion of water in human fetal lung tissue using DWI. A secondary objective was to determine the relationship of the ADCs in the fetal lung to menstrual age and total lung volume. Normal pregnant volunteers underwent MRI. Simple linear regression was performed with Pearson correlation coefficient. Inter- and intra-examiner bias and 95% limits of agreement (LOA) were determined. Forty-seven scans were performed at a mean of 29.2 weeks. The median coefficient of variation for ADC was 5.6%. No differences in ADC values were found between right and left lungs. Normally distributed ADC measurements were not significantly correlated with either total lung volume or menstrual age. The mean ADC value was 1.75. Mean intra-examiner bias was −0.15 and inter-examiner bias was 2.2. These findings suggest that ADC measurements of the fetal lung are reproducible between "blinded" examiners and are independent of menstrual age or lung volume.

An extremely short cervix as a risk factor for adverse pregnancy outcome

A sonographically diagnosed short cervix (SCX) is the most powerful predictor of preterm delivery. We investigated the frequency and clinical significance of IAI, AF MMP-8 >23 ng/mL, in a cohort of 47 asymptomatic women with a SCX (< or = 15 mm) between 14-24 weeks. We found IAI in 4.3% of patients. Among those with a negative AF culture, the prevalence of IAI was 22% and patients with a negative AF culture, but with IAI, had a higher rate of delivery within seven days (40% vs. 5.7%) and a shorter median diagnosis-to-delivery interval than those without IAI (18 vs. 42 days). Moreover, we investigated whether the risk of early spontaneous preterm delivery (sPTD) in asymptomatic women with a SCX changes as a function of gestational age at diagnosis. The cohort study included 109 asymptomatic singleton pregnancies with a SCX at 14-24 weeks; the patients were stratified by gestational age at diagnosis. Women diagnosed with a SCX at less than 20 weeks had a higher rate of sPTD at less than 28 weeks (77% vs. 31%) and at less than 32 weeks (81% vs. 48%), and a shorter median diagnosis-to-delivery interval (21 vs. 61.5 days) than those diagnosed at 20-24 weeks. The rate of amniotic fluid "sludge" was higher among patients diagnosed at less than 20 weeks than those diagnosed between 20-24 weeks (92% vs. 48%). We further evaluated the pregnancy outcome of asymptomatic patients with a non-measurable cervical length (0 mm) between 14-28 weeks. This retrospective cohort study included 78 patients with singleton pregnancies. We found that in 75% of the patients delivered at less than 32 weeks of gestation; the median diagnosis-to-delivery interval was 20.5 days, and the delivery rate within 7 and 14 days was 28% and 36%, respectively. Patients with a non-measurable cervix diagnosed at less than 24 weeks had a shorter median diagnosis-to-delivery interval than those diagnosed between 24-28 weeks (17.5 vs. 41 days). These studies demonstrate that a sonographic diagnosis of SCX in the midtrimester is a risk factor for preterm delivery and IAI and that almost 65% of asymptomatic women with a 0 mm cervix in the second trimester will not deliver within 2 weeks, yet 75% of them will deliver before 32 weeks of gestation.


  • Romero R, Velez Edwards DR, Kusanovic JP, Hassan SS, Mazaki-Tovi S, Vaisbuch E, Kim CJ, Chaiworapongsa T, Pearce BD, Friel LA, Bartlett J, Anant MK, Salisbury BA, Vovis GF, Lee MS, Gomez R, Behnke E, Oyarzun E, Tromp G, Williams SM, Menon R. Identification of fetal and maternal single nucleotide polymorphisms in candidate genes that predispose to spontaneous preterm labor with intact membranes. Am J Obstet Gynecol. 2010;202:431:e1-34.
  • Kim CJ, Romero R, Kusanovic JP, Yoo W, Dong Z, Topping V, Gotsch F, Yoon BH, Chi JG, Kim JS. The frequency, clinical significance, and pathological features of chronic chorioamnionitis: a lesion associated with spontaneous preterm birth. Mod Pathol. 2010;23:1000-1011.
  • DiGiulio DB, Romero R, Kusanovic JP, Gómez R, Kim CJ, Seok KS, Gotsch F, Mazaki-Tovi S, Vaisbuch E, Sanders K, Bik EM, Chaiworapongsa T, Oyarzún E, Relman DA. Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre-labor rupture of membranes. Am J Reprod Immunol. 2010;64:38-57.
  • Kusanovic JP, Romero R, Chaiworapongsa T, Erez O, Mittal P, Vaisbuch E, Mazaki-Tovi S, Gotsch F, Edwin SS, Gomez R, Yeo L, Conde-Agudelo A, Hassan SS. A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia. J Matern Fetal Neonatal Med. 2009;22:1021-1038.
  • Madsen-Bouterse SA, Romero R, Tarca AL, Kusanovic JP, Espinoza J, Kim CJ, Kim JS, Edwin SS, Gomez R, Draghici S. The transcriptome of the fetal inflammatory response syndrome. Am J Reprod Immunol. 2010;63:73-92.


  • Tinnakorn Chaiworapongsa, MD, Wayne State University School of Medicine, Detroit, MI
  • Russell Deter, MD, Baylor College of Medicine, Houston, TX
  • Daniel B DiGiulio, MD, Stanford University School of Medicine, Stanford, CA
  • Zhong Dong, MD, PhD, Wayne State University School of Medicine, Detroit, MI
  • Sorin Draghici, PhD, Wayne State University School of Medicine, Detroit, MI
  • Offer Erez, MD, 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
  • Juan Pedro Kusanovic, MD, Wayne State University School of Medicine, Detroit, MI
  • Michelle Lampl, MD, PhD, Department of Anthropology, Emory University, Atlanta, GA
  • Wesley Lee, MD, William Beaumont Hospital, Royal Oak, MI
  • Shali Mazaki-Tovi, MD, Wayne State University School of Medicine, Detroit, MI
  • Ramkumar Menon, PhD, Rollins School of Public Health of Emory University, Nashville, TN
  • Pooja Mittal, MD, Wayne State University School of Medicine, Detroit, MI
  • David Relman, MD, Stanford University School of Medicine, Stanford, CA
  • Wade Rogers, PhD, University of Pennsylvania School of Medicine, Philadelphia, PA
  • 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
  • Derek Wildman, PhD, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI
  • Scott Williams, PhD, Vanderbilt University, Nashville, TN
  • Lami Yeo, MD, Wayne State University School of Medicine, Detroit, MI
  • Bo Hyun Yoon, MD, PhD, Seoul National University, Seoul, Korea


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