Objective
MicroRNAs (miRNAs), which are highly conserved single-stranded noncoding RNAs that play a crucial role in gene regulation, have now been identified as important players in many diseases states. MiRNAs have also been demonstrated to be reliable and useful biomarkers to identify those women who are at risk for specific adverse outcomes. The objective of this study was to determine whether miRNA profiles in maternal blood are different in women who are destined to have a preterm, compared with a term, birth.
Study Design
A nested case-control study was performed with maternal serum that was collected as part of a larger prospective cohort. MiRNA expression profiles in maternal serum were compared between women who ultimately had a preterm birth (n = 40) compared with term birth (n = 40). MiRNA expression profiles were created with the use of the Affymetrix GeneChip miRNA Array. The data were analyzed with the significance of analysis of microarrays and principle components analyses. A false discovery rate of 20% was used to determine the most differentially expressed miRNAs.
Results
Of the 5640 miRNAs that were analyzed on the array, 4 miRNAs were significantly different between cases and control subjects. Two of the 4 miRNAs were mature miRNAs. The fold difference in expression was <2 for all 4 miRNAs.
Conclusion
MiRNA profiles in maternal blood were not significantly different in women who were destined to have a preterm, compared with a term, birth. MiRNAs in maternal blood are unlikely to become clinically useful biomarkers for the prediction of preterm birth.
Preterm birth remains the leading cause of childhood morbidity and death. Although there have been some clinical trials that have demonstrated a reduction in the preterm birth rate by targeting women at high risk, preventative or interventional strategies that significantly reduce the national and international preterm birth rate have not yet been realized. Although women with a previous preterm birth are at high risk for recurrent spontaneous preterm birth, most preterm births occur in women without this history. As such, accurate identification of those women who are at risk for preterm birth, weeks if not months, before the clinical event, would allow for improved use of resources (use of steroids, transfer to appropriate hospital facilities) as well as the potential for targeting novel therapeutics to prevent preterm birth.
Biomarkers have been used extensively in the medical field with notable success. The use of markers such as high-sensitivity c-reactive protein or prostate specific antigen have led to risk stratification algorithms for treatment options in the cardiovascular and cancer fields, respectively. Biomarker use in the field of preterm birth has been less successful. Although some studies demonstrate the utility of some biomarkers, most studies have revealed poor test characteristics for most proposed biomarkers, thus limiting any clinical utility. Based on available evidence that has demonstrated an association between inflammatory processes and preterm birth, much of the biomarker work in preterm birth has focused on inflammatory mediators. Reviews of these works report insufficient evidence for the use of any of these biomarkers in predicting preterm birth.
MicroRNAs (miRNA) may provide a new opportunity for biomarker discovery in the field of preterm birth. In the last decade, miRNA biology has emerged as an important player in both physiologic and pathophysiologic responses. MiRNAs are now implicated in varied disease states that include cancer and cardiovascular disease and are considered to be important therapeutic targets. MiRNAs can be released into the circulation and have been targeted as potential biomarkers for diverse disease states.
Our objective for this study was to determine whether there was a distinct miRNA profile in maternal blood collected from women who were destined to have a preterm birth compared with a term birth. For this study, we performed a nested case-control within a prospective cohort of women at high risk for preterm birth.
Materials and Methods
For this discovery work, we performed a nested case-control study with patients who were enrolled as part of prospective cohort study (The Biomarker Study, clinical trials NCT01148654 ) at the Hospital of the University of Pennsylvania. The study was approved (protocol # 807678) by the institutional review board. The parent study was a prospective cohort study that consisted of women with singleton pregnancies from 22-33 6/7 weeks’ gestational age who presented to the labor and delivery triage unit with complaints concerning preterm labor. Patients were excluded for multiple-gestation, major fetal anomaly, intrauterine fetal death, severe preeclampsia before enrollment, chronic steroid or immunosuppressive drug use, active immunologic disease, acute systemic febrile illness, and/or pregestational diabetes mellitus. Patients who were either not delivered at our institution or whose infants were transferred to a different hospital for care were also excluded from these analyses. Over the study period, 1067 women were enrolled. Of those women, 39% had a preterm birth at <37 weeks’ gestation.
Patients were enrolled in the study by trained clinical research coordinators who obtained informed consent at the time of enrollment. Patients were enrolled at any point during their assessment or admission for preterm labor. Therefore, some patients were enrolled at the time of admission to the hospital; others might have been enrolled after being admitted for treatment of preterm labor. For this study, 57% of women received betamethasone for fetal benefit. Considering enrollment into the study could occur at any point from presentation through admission, the timing of the maternal blood draw to betamethasone administration varied about the cases and control subjects. Once a patient was enrolled in the study, all management decisions were made by the treating physician according to the standard of care at our institution. Women were enrolled from April 2009 through March 2012. After informed consent was given, maternal blood was obtained by routine venipuncture. Specimens were processed to obtain serum. Serum samples were aliquoted and then immediately frozen. Samples were stored at –80°C until use.
For the study described herein, we identified all women who had a preterm birth at <37 weeks’ gestation. From that group of women, we further identified women who delivered at <30 weeks’ gestation to provide a more homogenous phenotype of preterm birth. Through detailed chart review, we confirmed that these preterm births were spontaneous and that gestational age at delivery was <30 weeks. Forty control subjects were then selected at random.
Data analyses
Pearson χ 2 analyses or Fisher exact, as appropriate, were used to determine associations between categoric demographic variables and preterm birth. Nonparametric comparisons that included Wilcoxon-Rank Sum tests were performed to assess associations between continuous variables and preterm birth. A probability value < .05 was considered statistically significant.
MiRNA profiling
Total RNA isolation
Total RNA, which included miRNA and other small RNAs, was isolated from human serum by phenol/chloroform extraction followed by column-based purification according to the manufacturer’s instructions (miRNeasy Serum/plasma kit; Qiagen, Valencia, CA). The RNA was eluted in 14 μL of RNase-free water. The RNA concentration was then determined with a spectrophotometer (Nanadrop 2000 Spectrophotometer; Nanodrop, Rockland, DE) to understand potential limitations of quantifying RNA in this manner from biofluids. RNA integrity was not measured because these samples are composed of extracellular RNA. The isolated RNA was then used for RNA profiling with the use of a miRNA array.
MiRNA array methods and analysis
MicroRNA profiling was performed on term (n = 40) and spontaneous preterm birth samples (n = 40). Total RNA (400 ng) was 3′ labeled with the use of the FlashTag Biotin HSR RNA Labeling Kit, per the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Samples were then hybridized onto Genechip miRNA 3.0 arrays for 18 hours (Affymetrix). Hybridization images were scanned and digitized with the Genechip Scanner 3000 (Affymetrix). The normalized signal intensity was log 2 transformed, and data analysis was performed with Partek Genomic Suite software (version 6.6; Copywrite, Partek Inc, St. Louis MO). Global sample variation was assessed by principle components analysis (PCA). Statistical analysis of microarrays ( http://www-stat.stanford.edu/∼tibs/SAM/ ) was used to analyze the data and generate false discovery rates (FDRs) for individual microRNAs. After using SAM, an FDR <20% was selected. MicroRNAs with a >2-fold expression change and FDR <20% were considered significantly altered.
Results
Clinical demographics
Maternal age and race were not significantly different between the cases and control subjects ( Table 1 ). As expected with our study design, gestational age at delivery was significantly different in cases and control subjects. As might be anticipated, more cases had a history of spontaneous preterm birth than the control subjects. The gestational age of blood draw was not significantly different between cases and control subjects.
| Demographic | Full term birth (n = 40) | Preterm birth (n = 40) | P value |
|---|---|---|---|
| Age, y a | 24.06 (21.05–28.85) | 24.04 (21.35–31.00) | .89 b |
| Black race, n (%) | 28 (70.00) | 35 (87.50) | .056 c |
| Previous preterm birth, n (%) | 2 (5.00) | 14 (35.00) | .003 c |
| Gestational age at delivery, wks a | 39.9 (39.6–40) | 27.3 (25.75–29.35) | < .001 b |
| Gestational age at blood draw, wks a | 26.75 (25.25–28.45) | 26.35 (24.4–28) | .23 b |
a Data are presented as median (interquartile range)
b Calculated by nonparametric comparison of medians
MiRNA array
The results of the PCA plot reveal that there is no clustering of women based on the array results ( Figure ). Of the 5640 miRNAs that were analyzed on the array, 4 microRNAs (stem-loop or mature) were altered between the preterm and term birth patients ( Table 2 ). MiR-200a* and miR-4695-5P were mature miRNA species, whereas miR-665 and miR-887 were detected in their stem-loop structure, which suggests that these last 2 species were not in their active form. All 4 targets had a <2-fold change in expression between preterm and term delivering women. All other targets had an FDR of ≥65% that suggests that there is no significant difference in expression between cases and control subjects.
