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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
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. 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, our emphasis was the study of fetal growth and the development of formulae to improve the estimation of fetal weight based on three-dimensional parameters.

Widespread microbial invasion of the chorioamniotic membranes is a consequence and not a cause of intra-amniotic infection

The pathway of microbial invasion leading to intra-amniotic infection has not been elucidated. The traditional view is that bacteria from the lower genital tract traverse the cervix and gain access to the decidua, generating a diffused infection of the decidua and membranes, and only after this, enter the amniotic cavity. This view was inconsistent with a number of observations made by our Branch in the recent past. Therefore, we conducted a series of studies to determine whether substantial bacterial propagation in the chorioamniotic membranes (CAMs) precedes microbial invasion of the amniotic cavity (MIAC). Bacteria (detected with a live/dead stain and FISH) were more frequently detected in the amniotic fluid (AF) than in the CAMs of patients with positive AF cultures for microorganisms. Bacteria were detected more frequently in the CAMs as chorioamnionitis worsened. A fraction of patients with chorioamnionitis (or MIAC) did not have bacteria in the CAMs. The median 16S rRNA gene copy number in the amnion was significantly greater than in the chorion. Bacteria were not detected in the CAMs or the AF of women at term without labor (this is in contrast to previous reports). Collectively, these findings indicate that MIAC does not follow widespread infection of CAMs, but precedes it: the initial stage is MIAC through a discrete region of the CAMs, followed by intra-amniotic proliferation; bacterial invasion of the CAMs primarily extends from the AF. This study emphasizes the importance of assessing the intra-amniotic compartment for diagnosis and treatment of preterm birth. It also stresses the need for non-invasive diagnosis of intra-amniotic infection by examining cervico-vaginal fluid.

A primate subfamily of galectins expressed at the maternal-fetal interface that promote immune cell death

Galectins are glycoproteins that regulate immune responses through the recognition of cell-surface glycans. Investigators at the PPRO found that 16 human galectin genes are expressed at the maternal-fetal interface and demonstrated that a cluster of 5 galectin genes on human chromosome 19 emerged during primate evolution. We showed that human placenta-specific galectins are predominantly expressed by the syncytiotrophoblast, a primary site of metabolic exchange where, during early pregnancy, the fetus comes into contact with immune cells circulating in maternal blood. Because ex vivo functional assays demonstrated that placenta-specific galectins induce the apoptosis of T lymphocytes, we proposed that these galectins reduce the danger of maternal immune attacks on the fetal semi-allograft, presumably conferring additional immune tolerance mechanisms, and in turn sustain hemochorial placentation during the long gestation of anthropoid primates.

Villitis of unknown etiology is associated with a distinct pattern of chemokine up-regulation in the feto-maternal and placental compartments: implications for conjoint maternal allograft rejection and maternal anti-fetal graft-versus-host disease

Villitis of unknown etiology (VUE) is a destructive inflammatory lesion of the placenta characterized by participation of Hofbauer cells and maternal T cells. The fundamental immunopathology of VUE is unknown. The PPRO conducted a series of studies to characterize the differences in the placental transcriptome between placentas with and without VUE and to determine whether VUE is associated with systemic maternal and/or fetal inflammatory responses. We found differential expression of 206 genes in VUE, and pathways over-represented were related to the immune response. The mRNA expression of a subset of chemokines and their receptors was higher in VUE placentas than in normal placentas (p<0.05). Blood cell mRNA showed a higher expression of CXCL9 and CXCL13 in the mother, and CXCL11 and CXCL13 in the fetus of VUE cases (p<0.05). We propose that VUE is a unique state combining maternal allograft rejection and maternal anti-fetal graft-vs-host disease mechanisms.

Expression patterns of microRNAs in the chorioamniotic membranes: a role for microRNAs in human pregnancy and parturition.

MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of gene expression during development. We conducted a series of studies to determine gestational age–dependent changes in miRNA expression in the chorioamniotic membranes and to assess the significance of miRNAs in human pregnancy and parturition. Using microarrays, we compared the expression profile of 455 miRNAs between patients at term without labor (TNL), in labor (TL), and preterm labor (PTL). A total of 39 miRNAs were differentially expressed between term and preterm cases (31 were down-regulated at term). The expression of ten miRNAs, including miR-338, was decreased at term. Computational analyses identified PLA2G4B, a phospholipase implicated in parturition, as a putative target of miR-338. Inhibition of endogenous miR-338 with anti-miR-338 increased the mRNA and protein expression of PLA2G4B in decidual cells (confirmed by luciferase assay with reporter constructs). Interestingly, the expression of Dicer, a key miRNA-processing enzyme, was markedly decreased in TL. These novel findings suggest that post-transcriptional regulation of genes by miRNAs plays a role in pregnancy and parturition.

A subset of patients destined to develop spontaneous preterm labor has an abnormal angiogenic/anti-angiogenic profile in maternal plasma: evidence in support of pathophysiologic heterogeneity of preterm labor derived from a longitudinal study

An imbalance between angiogenic and anti-angiogenic factors in maternal blood has been observed in several obstetrical syndromes, and in particular, preeclampsia. However, vascular lesions have been identified in a subset of patients with spontaneous preterm labor (PTL). We proposed that PTL may be one of the manifestations of an anti-angiogenic state. The PPRO conducted a number of studies to determine if patients destined to develop spontaneous PTL and delivery had changes in the profile of plasma concentrations of angiogenic and anti-angiogenic factors. Plasma sEng concentrations were higher in patients destined to develop preterm labor than those of normal pregnant women from 15-20 weeks onward. The difference became statistically significant at 28 weeks, or approximately 5-10 weeks prior to the diagnosis of PTL. This observation has implications for the understanding of the mechanisms of disease responsible for the preterm parturition syndrome.

Isobaric labeling and tandem mass spectrometry: a novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation

The PPRO has used high-dimensional biology techniques to understand the mechanisms of disease for preterm labor and to identify potential biomarkers. We used, for the first time, the combination of Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) labeling and tandem mass spectrometry to identify proteins differentially expressed in AF samples of women with PTL with and without intra-amniotic infection/inflammation (IAI). We identified many novel proteins that were up-regulated in AF of patients with PTL and IAI. Moreover, we also found differential expression of proteins in patients who delivered preterm in the absence of IAI compared with those who delivered at term. We employed gene ontology enrichment analysis to reveal families of proteins participating in distinct biological processes, including host defense, anti-apoptosis, metabolism/catabolism and cell and protein mobility, localization, and targeting. Using iTRAQ, we discovered 82 proteins differentially expressed in three clinical subgroups of PTL, 67 of which were hitherto unknown. Of particular importance is the identification of proteins differentially expressed in AF of women who delivered preterm in the absence of IAI. This is the first report of the positive identification of biomarkers in this subgroup of patients.

Fractional limb volume—a soft tissue parameter of fetal body composition: validation, technical considerations and normal ranges during pregnancy

Three-dimensional ultrasonography (3DUS) allows evaluation of the soft tissue of fetal limbs, a parameter that has been described for the evaluation of fetal nutritional status. The fractional limb volume addresses the technical limitations of fetal weight estimation and growth assessment. Our group's goal was to provide normal reference ranges for fractional limb volume as a new index of the fetal nutritional status. We also assessed the reproducibility of fractional fetal limb volume measurements during the second and third trimesters of pregnancy. A prospective, cross-sectional study of 387 gravid women between 18.0 and 42.1 weeks of gestation was conducted. Slices were traced manually to obtain fractional arm (AVol) or fractional thigh (TVol) volume and reference charts were established. No gender differences were found in these soft tissue measurements. Intra-observer mean bias for fractional limb volumes were: 2.2 ± 4.2% for AVol and 2.0 ± 4.2% for TVol. Inter-observer bias and agreement were -1.9 ± 4.9% for AVol and -2.0 ± 5.4% for TVol. Fractional limb volume assessment may improve the detection and monitoring of malnourished fetuses because this soft tissue parameter can be obtained quickly and reproducibly during mid to late pregnancy.

Fetal growth parameters and birth weight: their relationship to neonatal body composition

Air displacement plethysmography is a non-invasive technique that uses total body volume and mass to derive body composition, including percentage body fat (%BF) and lean body mass (%LBM). This technology has recently been applied to assess neonatal and infant body composition. Investigators at the PPRO studied prospectively the relationship between standard fetal biometry and soft tissue parameters with birth weight in third-trimester pregnancies using three-dimensional ultrasonography (3DUS). Soft tissue parameters included AVol and TVol. The TVol had the strongest correlation with newborn %BF of all single-parameter models. This parameter alone explained 46.1% of the variability in %BF. Birth weight similarly explained 44.7% of the variation in %BF. Abdominal circumference (AC) and estimated fetal weight (EFW) accounted for only 24.8% and 30.4% of the variance in %BF, respectively. Growth parameters, such as femoral diaphysis length (FDL) (14.2%), head circumference (HC) (7.9%) and biparietal diameter (BPD) (4.0%), contributed the least towards explaining the variance in %BF. TVol accounts for a large proportion of the variance in neonatal percentage body fat compared with traditional parameters measured during routine sonography such as abdominal circumference or estimated fetal weight.

Downward percentile crossing as an indicator of an adverse prenatal environment

Postnatal health sequelae of low birth weight have been attributed to “poor fetal growth” secondary to adverse prenatal environments; identification of prenatal growth-restricting events is essential to clarify pathways and mechanisms of fetal growth. We previously reported that patients with an episode of preterm labor which subsided and led to a term delivery were associated with an excess risk of giving birth to small-for-gestational-age (SGA) neonates. The PPRO conducted a study to examine whether an episode of preterm labor was associated with compromised fetal growth. Fetal size at the end of the second trimester and at birth were compared among women with normal pregnancies and those who experienced an episode of preterm labor (<37 weeks) and subsequently delivered at term (≥37 weeks). Longitudinal estimated fetal weight and changes in weight standard scores across the third trimester were studied. Neonates delivered at term after an episode of preterm labor were smaller at birth relative to their peers than at the end of the second trimester, and were 47% more likely to have experienced clinically significant downward centile crossing than their peers. We concluded that an episode of preterm labor may represent evidence of an adverse prenatal environment. However, such an insult is not sufficient to cause irreversible preterm labor and delivery.

Growth perturbations in a phenotype with rapid fetal growth preceding preterm labor and term birth

The PPRO conducted a retrospective analysis of fetal biometry assessed by serial ultrasound in a prospective cohort of pregnant women to study fetal growth patterns predating an episode of preterm labor. Fetal growth patterns among uncomplicated pregnancies and those with an episode of preterm labor followed by term delivery were examined across time intervals (16-22 weeks, 22-28 weeks, and 28-34 weeks) using multilevel mixed-effects regression analysis. Fetal weight growth rate increased faster at 16 weeks among pregnancies with an episode of preterm labor, declined across midgestation (22-28 weeks), and rebounded between 28 and 34 weeks. This was associated with changes in AC growth and proportionately larger BPD from 22 weeks, greater femur, BPD and HC dimensions relative to AC across midgestation (22-28 weeks), followed by proportionately smaller FDL, and subsequently, BPD. A distinctive “rapid growth phenotype” characterized fetal growth preceding an episode of preterm labor among patients who delivered at term. Perturbations in AC growth and patterns of proportionality suggest that altered fetal growth precedes an episode of preterm labor.

Early rapid growth, early birth: accelerated fetal growth and spontaneous late preterm birth

The past two decades in the United States have seen a 24% rise in spontaneous late preterm delivery (34.0 to 36.9 weeks) of unknown etiology. This study was conducted to compare fetal growth between patients with spontaneous late preterm birth and those who delivered at term in a cohort of pregnancies followed longitudinally. Preterm-delivered fetuses were significantly larger than their term-delivered peers by mid-second trimester in estimated fetal weight, head, limb, and abdominal dimensions, and they followed different growth trajectories. Piecewise regression assessed time-specific differences in growth rates at 4-week intervals from 16 weeks. EFW and AC growth rates slowed at 20 weeks among the preterm-delivered, only to match and/or exceed their term-delivered peers at 24-28 weeks. Growth rates predicted birth timing: one standard score of EFW increased the odds ratio for late preterm birth from 2.8 prior to 23 weeks, to 3.6 between 23 and 27 weeks. After 27 weeks, increasing size was protective. Our study showed, for the first time, that a distinctive fetal growth pattern across gestation precedes spontaneous late preterm birth and stressed the importance of mid-gestation for the generation of fetal growth disorders.

Publications

Kim MJ, Romero R, Gervasi MT, Kim JS, Yoo W, Lee DC, Mittal P, Erez O, Kusanovic JP, Hassan SS, Kim CJ. Widespread microbial invasion of the chorioamniotic membranes is a consequence and not a cause of intra-amniotic infection. Lab Invest 2009 89:924-936.
Than NG, Romero R, Goodman M, Weckle A, Xing J, Dong Z, Xu Y, Tarquini F, Szilagyi A, Gal P, Hou Z, Tarca AL, Kim CJ, Kim JS, Haidarian S, Uddin M, Bohn H, Benirschke K, Santolaya-Forgas J, Grossman LI, Erez O, Hassan SS, Zavodsky P, Papp Z, Wildman DE. A primate subfamily of galectins expressed at the maternal-fetal interface that promote immune cell death. Proc Natl Acad Sci USA 2009 106:9731-9736.
Romero R, Kusanovic JP, Gotsch F, Erez O, Vaisbuch E, Mazaki-Tovi S, Moser AR, Tam S, Leszyk J, Master SR, Juhasz P, Pacora P, Ogge G, Gomez R, Yoon BH, Yeo L, Hassan SS, Rogers W. Isobaric labeling and tandem mass spectrometry: a novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation. J Matern Fetal Neonatal Med 2009 6:1-20.
Lee W, Balasubramaniam M, Deter RL, Hassan SS, Gotsch F, Kusanovic JP, Goncalves LF, Romero R. Fractional limb volume: a soft tissue parameter of fetal body composition, validation, technical considerations and normal ranges during pregnancy. Ultrasound Obstet Gynecol 2009 33:427-440.
Lampl M, Kusanovic JP, Erez O, Gotsch F, Espinoza J, Goncalves L, Lee W, Gomez R, Nien JK, Frongillo EA, Romero R. Growth pertubations in a phenotype with rapid fetal growth preceding preterm labor and term birth. Am J Hum Biol 2009 21:782-792.

Collaborators

Emmanuel Bujold, MD, University of Montreal, Quebec, Canada
Tinnakorn Chaiworapongsa, MD, Wayne State University School of Medicine, Detroit, MI
Russell Deter, MD, Baylor College of Medicine, Houston, TX
Zhong Dong, MD, PhD, Wayne State University School of Medicine, Detroit, MI
Offer Erez, MD, Wayne State University School of Medicine, Detroit, MI
Morris Goodman, PhD, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI
Lawrence Grossman, PhD, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI
Yu Mi Han, PhD, Center for Molecular Medicine and Genetics, 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
Mi Jeong Kim, 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
Pooja Mittal, MD, Wayne State University School of Medicine, Detroit, MI
Daniel Montenegro, BS, Wayne State University School of Medicine, Detroit, MI
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
Lami Yeo, MD, Wayne State University School of Medicine, Detroit, MI
Bo Hyun Yoon, MD, PhD, Seoul National University, Seoul, Korea

Contact

For more information, email romeror@mail.nih.gov.

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