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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

Preterm birth is the leading cause of perinatal morbidity and mortality worldwide. The Perinatology Research Branch proposed that preterm parturition is a syndrome caused by multiple pathologic processes, one of which is an untimely decline in progesterone action, manifested by "silent" shortening of the uterine cervix. Previous work conducted by our Program showed that asymptomatic women who have a cervical length less than 15mm in the second trimester of pregnancy have a 50% likelihood of delivering an early preterm neonate. However, prediction of preterm birth needs to be accompanied by a strategy to reduce the frequency of this complication. In a previous randomized clinical trial of vaginal progesterone vs. placebo in women with a short cervix, we reported that treatment was associated with a 45% reduction in the rate of preterm birth (less than 33 weeks of gestation), and a decrease in the rate of respiratory distress syndrome, the most common complication of prematurity. This year, we conducted studies to examine the relative merits of vaginal progesterone vs. cervical cerclage in patients with a short cervix and a prior history of preterm birth and found that medical treatment is as effective as the surgical approach. We also identified a novel pathologic finding associated with preterm labor without infection: chronic chorioamnionitis, which is likely attributable to maternal anti-fetal rejection, and we described a novel form of systemic fetal inflammation.

The Program also studies other great obstetrical syndromes that account for the high rate of infant mortality in the United States including preeclampsia, fetal growth restriction, fetal death, clinical chorioamnionitis, and meconium aspiration syndrome.

Congenital anomalies continue to be a leading cause of perinatal mortality in the United States. Congenital heart disease is the leading organ-specific birth defect, as well as the leading cause of infant mortality from congenital malformations. The lack of prenatal identification of congenital heart defects can have adverse consequences for the neonate. More than half the infants affected with congenital heart disease are born to mothers without any previously known risk factors, which provides the impetus to perform a comprehensive screening examination of the fetal heart in all pregnancies. Yet, the prenatal diagnosis of congenital heart disease remains a challenge, as the sensitivity of ultrasound has ranged from 15–39%. Examination of the fetal heart is time-consuming, and expertise and skill are required. Therefore, the examination frequently does not include all the standard recommended cardiac views. Spatiotemporal image correlation (STIC) technology allows the acquisition of a volume dataset from the fetal heart, and displays a cine loop of a complete single cardiac cycle in motion. A growing body of evidence suggests that 4-D sonography with STIC facilitates examination of the fetal heart. However, extracting and displaying the recommended diagnostic planes from a volume dataset that can be dissected in many ways (i.e., planes) requires an in-depth knowledge of anatomy and is difficult and operator-dependent. After several years of work, we developed a novel method for visualizing standard fetal echocardiography views from volume datasets obtained with STIC and application of "intelligent navigation" technology. We also use magnetic resonance imaging for the study of fetal brain neuroconnectivity.

A novel molecular microbiologic technique for the diagnosis of microbial invasion of the amniotic cavity and intra-amniotic infection

One of every three preterm births occurs to a mother with intra-amniotic infection, which is largely subclinical. The standard method for the diagnosis of infection is cultivation of amniotic fluid to detect bacteria. However, limitations of culture methods include the time required (days) to identify microorganisms and the fact that many microbes are difficult to culture. Molecular detection of microorganisms is an increasingly important diagnostic tool; it can be done with either broad-range PCR methods aimed at the 16S rRNA gene conserved in bacterial species or with specific PCR assays. However, until recently, the techniques have remained research procedures, because the time required to obtain results have precluded their use as a point-of-care test. This year, our Branch reported a new method that allows molecular identification of the genus and species of bacteria and a large number of viruses within eight hours and that provides an estimate of the microbial burden. The method uses a combination of PCR and electron spray ionization mass spectrometry (ESI-MS) to identify and quantify genomic material. We applied the technology to diagnose intra-amniotic infection in patients with preterm labor and intact membranes and found that more infections were detected with PCR ESI-MS than with cultivation techniques. We identified a threshold (>17 genome equivalents/well) above which a positive result was associated with intra-amniotic inflammation, acute histologic inflammatory lesions of the placenta, a short interval to delivery, and a higher rate of perinatal morbidity. The results indicate that rapid diagnosis of intra-amniotic infection is now possible, opening the door for timely and specific interventions to reduce morbidity owing to infection-induced preterm birth.

The lower genital tract as a source for intra-amniotic infection

A central question has been why some women develop an ascending intra-amniotic infection while most do not. The relationship between the mucosa of the lower genital tract (vagina and cervix) and the microbial ecosystem appears to be a key factor predisposing to ascending infection. Bacterial vaginosis—a change in the microbial ecosystem in which there is proliferation of anaerobic bacteria—confers risk for intra-amniotic infection and spontaneous preterm delivery. Similarly, Trichomonas vaginalis infection is a risk factor for preterm delivery. However, antibiotic treatment of asymptomatic women with bacterial vaginosis or infected with Trichomonas vaginalis has not reduced the rate of preterm delivery. A comprehensive understanding of microbial ecology, genetic factors that control susceptibility to infection and the inflammatory response is required, particularly in light of evidence that gene–environment interactions may predispose to preterm labor. Our Branch has conducted studies about the composition of the microbiota of the vagina in normal pregnant women and in those who subsequently delivered after spontaneous preterm labor.

The composition and stability of the vaginal microbiota of normal pregnant women

Characterization of the microbial composition of ecological niches in the human body, including the vagina, using culture-independent techniques, is now possible. This year, we reported a study that showed that the composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women. We studied non-pregnant women (N=32) and pregnant women who delivered at term (38-42 weeks) without complications (n= 22). Serial samples of vaginal fluid were collected from both non-pregnant and pregnant patients. A 16S rRNA gene sequence–based survey was conducted using pyrosequencing to characterize the structure and stability of the vaginal microbiota. Linear mixed-effects models and generalized estimating equations were used to identify the phylotypes whose relative abundance differed between the two study groups. The vaginal microbiota of normal pregnant women was different from that of non-pregnant women (higher abundance of Lactobacillus vaginalis, L. crispatus, L. gasseri, and L. jensenii and lower abundance of 22 other phylotypes in pregnant women). Bacterial community state type (CST) IV-B or CST IV-A characterized by high relative abundance of species of genus Atopobium, as well as the presence of Prevotella, Sneathia, Gardnerella, Ruminococcaceae, Parvimonas, Mobiluncus and other taxa previously shown to be associated with bacterial vaginosis, were less frequent in normal pregnancy. The stability of the vaginal microbiota of pregnant women was higher than that of non-pregnant women; however, during normal pregnancy, bacterial communities shift almost exclusively from community-state type dominated by Lactobacillus spp. to another community-state type dominated by different Lactobacillus spp. Thus, there are some changes in dynamics, but never from a community state type dominated by a Lactobacillus spp. to a community state lacking Lactobacillus spp.

The vaginal microbiota of pregnant women who subsequently have spontaneous preterm labor and delivery and those with a normal delivery at term

We then conducted a study to determine whether the vaginal microbiota of pregnant women who subsequently had a spontaneous preterm delivery is different from that of women who had a term delivery. We included a control group of pregnant women who had a term delivery and those who had a spontaneous preterm delivery before 34 weeks of gestation. Samples of vaginal fluid were collected longitudinally and stored at −70°C until assayed. A microbial survey using pyrosequencing of V1–V3 regions of 16S rRNA genes was performed, and we tested the hypothesis that the relative abundance of individual microbial species (phylotypes) was different between women who had a term vs. preterm delivery. We reported that the composition of the vaginal microbiota during normal pregnancy changed as a function of gestational age, with an increase in the relative abundance of four Lactobacillus spp. and decreased in anaerobe or strict-anaerobe microbial species as pregnancy progressed; however, no change in the relative abundance of bacterial taxa was observed between women who had a spontaneous preterm delivery and those who delivered at term. The same was the case for the frequency of the vaginal community state types. These early findings suggest that changes in the vaginal microbiota are not easily detected in women who subsequently have a spontaneous preterm delivery. The studies included both women whose placentas had evidence of acute histologic chorioamnionitis and those without such inflammatory lesions. Future studies will need to take into consideration the indices of the maternal immune response and functional aspects of the vaginal microbiota.

Bacteria and endotoxin in meconium-stained amniotic fluid

Meconium-stained amniotic fluid (MSAF) affects 5–20% of all pregnancies (400,000–600,000 deliveries per year in the United States alone) and is a risk factor for meconium aspiration syndrome (MAS); however, only 5% of infants with MSAF develop MAS. A critical question is why some neonates exposed to meconium develop this syndrome while others do not. Attempts to prevent MAS with mechanical methods such as oropharyngeal, nasopharyngeal, and tracheal suctioning and amnio-infusion have been attempted, but none have proven effective. Patients with MSAF are at increased risk for clinical chorioamnionitis, puerperal endometritis, neonatal sepsis, and intra-amniotic infection. Therefore, we conducted a series of studies to determine whether MSAF was associated with the presence of bacteria in the amniotic fluid or bacterial products such as endotoxin. We found that 19% of patients with MSAF have microorganisms detected by culture, frequently encountering Gram-negative bacteria. Endotoxin was detected in 46% of patients with MSAF, and these patients were also more likely to have intra-amniotic inflammation (an elevation in AF IL-6). In a separate study, we found that MSAF containing bacterial endotoxin also had a higher concentration of secreted phospholipase A2, an enzyme implicated in the pathogenesis of MAS. Studies are now in progress to determine whether fetal systemic inflammation is a predisposing factor for the development of MAS.

Transdermal nitroglycerin for the treatment of preterm labor

The mainstay for the treatment of preterm labor remains arresting uterine contractility (tocolysis). We previously reported systematic reviews and meta-analyses of calcium-channel blockers for this indication. This year, we reported a systematic review and meta-analysis of transdermal nitroglycerin for the treatment of preterm labor. We found that, although transdermal nitroglycerin appears to be more effective than beta adrenergic receptor agonists, the current evidence does not support its routine use as a tocolytic agent for the treatment of preterm labor.

MR venography of the fetal brain using susceptibility weighted imaging

Susceptibility weighted imaging (SWI) is an MR technique invented by our group that uses T2* weighted magnitude and phase images to improve visualization of small veins and microhemorrhages. SWI also allows quantitative assessment of blood oxygenation, using principles of quantitative susceptibility mapping. For the first time, we demonstrated that the fetal brain venous system could be imaged using SWI/MRI. The cerebral venous vasculature was visible in 86.4% of cases. The image quality was scored as being of diagnostic quality (68.2%), diagnostic quality with minor artifacts (22.7%), and non-diagnostic (9.1%). We have thus shown that MRI/SWI are of value in the differential diagnosis of hemorrhage vs. calcification in fetuses with brain lesions, which is not possible with any other diagnostic technique.

Ultrasound elastography: the relationship between results of elastography of the uterine cervix and spontaneous preterm delivery

Patients with a sonographically detected short cervix are at increased risk for preterm delivery, and vaginal progesterone decreases the rate of early preterm birth by 45%. However, a fraction of patients with a short cervix do not deliver preterm, and therefore, do not need treatment (either surgical or medical); at present, such patients cannot be identified. Ultrasound-derived elastography can be used to evaluate stiffness of tissue and is based on the estimation of tissue displacement (strain) within a defined region of interest when oscillatory compression is applied. We reported a study in which strain estimations were prospectively determined in 262 women (8–40 weeks of gestation). Mean strain rates were 14% and 5% greater among parous women with and without a history of PTD, respectively (vs. nulliparous women), and were 13% greater among women with a cervical length of 25–30 mm (vs. over 30 mm). Cervical tissue strain was more strongly associated with cervical length than gestational age. To determine whether there was an association between cervical strain and spontaneous preterm delivery, elastography was performed in 189 women from 16–24 weeks of gestation. Low strain values in the internal os were significantly associated with a lower risk of spontaneous PTD at less than 37 weeks of gestation. Women with strain values in or less than the 25th percentile (representing stiff tissue) in the endocervical canal and in the entire cervix were 80% less likely to have a spontaneous PTD than those with strain values above the 25th percentile. Therefore, ultrasound-based elastography can serve as a tool to identify patients with a short cervix who may not require treatment. Further studies will focus on the use of shear-wave elastography, which is superior to conventional elastography because the generation of the mechanical impulse that induces tissue displacement is operator-independent.

Human fetal brain neuroconnectivity

A major objective for neuroscience is to build a complete diagram of brain connections. Although this is being pursued in adults and children and is considered a high priority for the U.S. (Human Brain Project), little is known about functional neuroconnectivity during human fetal life. By leveraging the characterization of low-frequency intrinsic fluctuations in the blood oxygen level–dependent (BOLD) signals, functional connectivity MRI (fcMRI) provides information about macroscale brain organization. The approach is useful for determining emerging neural circuits in the human fetus, important because many neurodevelopmental disorders, such as autism spectrum disorders, learning disabilities and cerebral palsy, may result from disorders of neuroconnectivity. Building on our pioneering studies on fetal neuroconnectivity (Thomason M et al., Sci Trans Med 2013;5:173ra24), we applied graph theoretical analysis to gain insight into human fetal brain connectivity. Utilizing resting-state functional magnetic resonance imaging (fMRI) data from 33 healthy human fetuses at 19 to 39 weeks of gestational age (GA), our analyses revealed that the human fetal brain has modular organization and that such modules overlap with functional systems observed in the neonatal period. Age-related differences between younger (GA less than 31 weeks) and older (GA more than 31 weeks) fetuses demonstrate that brain modularity declines and that connectivity of the posterior cingulate to other brain networks becomes more negative with advancing GA. The results suggest that the human fetus has the capacity for information processing well before birth.

Publications

1. Romero R, Miranda J, Chaiworapongsa T, Chaemsaithong P, Gotsch F, Dong Z, Ahmed AI, Yoon B, Hassan SS, Kim C, Korzeniewski SJ, Yeo L, Kim Y. Sterile intra-amniotic inflammation in asymptomatic patients with a sonographic short cervix. J Matern Fetal Neonatal Med 2014;E-pub ahead of print.
2. Romero R, Hassan SS, Gajer P, Tarca AL, Fadrosh DW, Nikita L, Galuppi M, Lamont RF, Chaemsaithong P, Miranda J, Chaiworapongsa T, Ravel J. The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women. Microbiome 2014;2:4.
3. Romero R, Dey SK, Fisher SJ. Preterm labor: one syndrome, many causes. Science 2014;345:760-765.
4. Romero R, Miranda J, Chaemsaithong P, Kusanovic JP, Chaiworapongsa T, Dong Z, Ahmed AI, Shaman M, Lannaman K, Yoon BH, Hassan SS, Kim CJ, Korzeniewski SJ, Yeo L, Kim YM. Sterile and microbial-associated intra-amniotic inflammation in preterm prelabor rupture of membranes. J Matern Fetal Neonatal Med 2014;E-pub ahead of print.
5. Romero R, Yeo L, Chaemsaithong P, Chaiworapongsa T, Hassan SS. Progesterone to prevent spontaneous preterm birth. Semin Fetal Neonatal Med 2014;19:15-26.

Collaborators

• Tinnakorn Chaiworapongsa, MD, Wayne State University School of Medicine, Detroit, MI
• Agustin Conde-Agudelo, MD, Wayne State University School of Medicine, Detroit, MI
• Sonia Hassan, MD, Wayne State University School of Medicine, Detroit, MI
• Edgar Hernandez-Andrade, MD, Wayne State University School of Medicine, Detroit, MI
• Steven J. Korzeniewski, PhD, MSc, MA, Wayne State University School of Medicine, Detroit, MI
• Moriah Thomason, PhD, Wayne State University School of Medicine, Detroit, MI
• Lami Yeo, MD, Wayne State University School of Medicine, Detroit, MI

Contact

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

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