Functional Genomics and Proteomics in Term and Preterm Parturition

Roberto Romero, Helena Kuivaniemi and Gerard Tromp

Perinatology Research Branch, Intramural Division, National Institute of Child Health and Human Development (R.R.), Bethesda, Maryland 20892; and Center for Molecular Medicine and Genetics (H.K., G.T.) and Department of Surgery (H.K.), Wayne State University School of Medicine, Detroit, Michigan 48201

Address all correspondence and requests for reprints to: Roberto Romero, M.D., Perinatology Research Branch, National Institute of Child Health and Human Development, 4707 St. Antoine Boulevard, Detroit, Michigan 48201. E-mail: . warfiela{at}mail.nih.gov

Few biological processes as central to the survival of a species as parturition are so incompletely understood. Untimely birth due to the premature or delayed onset of labor is associated with an increased risk of perinatal death and long-term handicap. Even when labor begins at term, "failure to progress" happens so often that nowadays approximately one in every five births occurs by cesarean section. This is not due to disproportion between the size of the fetus and the maternal pelvis, because a substantial proportion of patients who are delivered by cesarean section with the indication of failure to progress during labor do, in fact, deliver vaginally a larger baby in subsequent pregnancies. Thus, it is likely that failure to progress represents a group of functional disorders (1) whose etiology remains to be determined.

Although considerable work has been done and the understanding of parturition has improved in the last two decades (2, 3, 4, 5, 6), much remains to be elucidated. Functional genomics, proteomics, and systems biology (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) hold the promise of allowing a comprehensive, systematic, and unbiased description of the pathways involved in the process of normal or abnormal parturition. Chan et al. (20) report in this issue of JCEM the use of suppression subtractive hybridization (SSH) (21) to identify genes differentially regulated in human myometrium during labor. Thus, functional genomics has arrived to the study of parturition, as confirmed by a few pioneering reports, many of which are in abstract form at the time of this writing (20, 22, 23, 24, 25, 26, 27, 28, 29, 30). Table 1Go describes the key features of such studies. Fundamental differences among them include the species (mice, rats, sheep, human), tissues examined (myometrium, fetal membranes), and methodology [SSH, microarray, differential-display PCR (DD-PCR)] (14), as well as whether confirmation of positive results was undertaken. Yet, the unifying theme is that all investigators, using either restricted or unrestricted approaches (also known as open- and closed-ended systems (14), have reported genes differentially regulated during parturition (20, 22, 23, 24, 25, 26, 27, 28, 29, 30).


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Table 1. Summary on functional genomics studies in pregnancy and parturition

 
Of the studies published to date, only two have examined changes in myometrium gene expression from women in spontaneous labor at term. Aguan et al. (25), using macroarrays for 588 genes and RNA isolated from the myometrium of three patients in spontaneous labor and from three patients not in labor, noted up- and down-regulation (defined as a change of 2-fold or greater) of 21 genes involved in a wide range of physiological processes, including smooth muscle contraction and relaxation, regulation of DNA metabolism, as well as transcriptional and cell cycle regulation. The study by Chan et al. (20) constitutes a major effort to use an unrestricted approach to identify differentially regulated genes during spontaneous labor at term. SSH was used with cDNA libraries constructed from the myometrium of one patient not in labor and a patient who underwent a cesarean section because of failure to progress in labor. Dot-blot screening of 400 positive clones indicated that 14 genes were up-regulated and 16 were down-regulated. Up-regulated genes included those encoding for proteins implicated in the mechanism of parturition in the pregenomic era [oxytocin receptor (31, 32), matrix metalloproteinase-9 (MMP-9) (33, 34, 35), fibronectin (36), and IL-8 (37, 38, 39, 40, 41, 42)], those not previously implicated (see Table 2Go), and four genes with no matching sequences in available databases. Northern blot analysis was performed for six genes and quantitative-RT-PCR (Q-RT-PCR) for three [IL-8, Mn superoxidase dismutase (MnSOD), and cyclophilin] in a set of samples from a larger cohort. The major findings were that 1) these three genes were up-regulated during spontaneous labor; 2) there were topographic differences in the expression of MnSOD in the lower uterine segment and fundal myometrium, but not for IL-8; and 3) IL-8 expression was higher in spontaneous labor than in induced labor.


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Table 2. Differentially expressed genes in the myometrium of women in labor

 
Notably, the results with the unrestricted approach (SSH) and the restricted approach (macroarrays) did not yield the same results. Table 2Go describes the genes found to be up-regulated in the two studies.

Why a difference? One explanation is that a restricted approach (macroarrays) is limited to probing the genes in the array. Of the 10 known genes found to be up-regulated by SSH, only one (IL-8) was present in the macroarray, and it was not detected as differentially regulated. Because Q-RT-PCR, as well as protein analysis in several studies (37, 38, 39, 40, 41, 42), indicates that IL-8 is up-regulated during labor, it seems that this observation represents a false negative result of the array experiment. The reasons that SSH did not identify up-regulated genes detected by the macroarray are less obvious. SSH is generally better suited for the identification of sizable differences (about 5-fold or greater) in rare transcripts, whereas arrays can detect smaller differences, although not necessarily of such rare transcripts (21). Thus, if the goal is a comprehensive detection of even small differences in the transcriptome, the two techniques are complementary. It may be prudent not to take at face value the results of any screening technique until they are confirmed with an independent methodology (e.g. Northern or Q-RT-PCR).

Two reports have used functional genomics to study the fetal membranes of patients who delivered preterm. Tashima et al. (23) applied SSH to identify differentially regulated genes in patients with preterm premature rupture of membranes (a condition that accounts for 40% of all preterm deliveries), whereas Marvin et al. (30) used macroarrays with 384 genes and focused on preterm birth associated with intrauterine infection. Tashima et al. (23) constructed cDNA libraries from the membranes of one patient who underwent a cesarean section at 28 wk for fetal distress and from three patients with preterm premature rupture of membranes at 25, 33, and 35 wk, of whom one had histological chorioamnionitis. Eight genes were found to be up-regulated, of which one was a false positive after Northern analysis. The genes whose differential regulation were confirmed are listed here with Locus symbols and LocusLink identifiers: IL-8 (IL8; 3576), complement factor B (BF; 629), ferritin (FTH1; 2495), the invariant gamma chain of class II histocompatibility leukocyte antigen (CD74; 972), F-actin capping protein ß-subunit (CAP2B; 832), chitinase precursor (CHI3L2; 1117), and a regulator of G protein signaling (RGS2; 15997). Marvin et al. (30) found that chemokines (including IL-8) and proinflammatory cytokines were also up-regulated in patients with infection-associated preterm delivery.

These exploratory investigations have confirmed the involvement of some molecules that have been considered important in the mechanism of parturition for some time and, more importantly, identified a series of new candidate genes that seem to be involved in normal or abnormal parturition. Among those that have already been implicated are chemokines such as IL-8 (37, 38, 39, 40, 41, 42), matrix degrading enzymes that are up-regulated during inflammation such as MMP-9 (33, 34, 35), and the oxytocin receptor (31, 32). Novel genes provide interesting leads, but the challenge will be to decide which ones to pursue. Clearly, the studies like those of Chan et al. (20) are a step forward in the elucidation of the molecular events involved in labor. Yet, much still needs to be learned about the comprehensive list of differentially regulated genes of various tissues in labor disorders, the consequences on the proteome and metabolome, and, more importantly, the significance in the physiology of parturition (parturition physiome).

Future studies will require a systematic examination of differentially regulated genes and proteins in maternal and fetal tissues involved in parturition (amnion, chorion, decidua, myometrium, cervix, etc.). It is necessary to standardize the format used for the communication of results. For example, we encourage investigators to report a list of all the differentially regulated genes with LocusLink and GenBank accession numbers. Otherwise, to compare the results from one study to another will be difficult (12, 43, 44, 45). A precise description of phenotype and experimental conditions, including information about pharmacological exposure in human studies, is highly desirable (12, 43, 44, 45).

Insofar as biological implications, the results from functional genomics studies (20, 22, 23, 24, 25, 26, 27, 28, 29, 30) support the hypothesis that parturition is an inflammatory-like process, because many of the up-regulated genes are physiologically linked to the control of inflammation (39, 40, 42, 46). To develop new therapeutic interventions, it will be necessary to distinguish between differentially regulated genes that are causally related to parturition and those that are merely associated with it. The study by Chan et al. (20), like other reports of biological phenomena in the postgenomic era (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), reveals the complexity of biological processes, including parturition. A gene-by-gene approach is likely to be inadequate in accurately describing such a complicated and heterogeneous process, as well as classifying labor abnormalities. High-throughput methods (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), able to provide a global overview, will be necessary. Only by classifying of these complex and heterogeneous disorders can we begin to develop a rational approach for the diagnosis, treatment, and prevention of labor disorders.

Acknowledgments

Footnotes

Abbreviations: DD-PCR, Differential-display PCR; MMP-9, matrix metalloproteinase-9; MnSOD, Mn superoxidase dismutase; Q-RT-PCR, quantitative-RT-PCR; SSH, suppression subtractive hybridization.

Received April 15, 2002.

Accepted April 16, 2002.

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