Objective
We sought to determine if second-trimester amniotic fluid thrombin-antithrombin (TAT) complexes concentration correlates with subsequent preterm birth.
Study Design
A cohort of 550 women with singleton nonanomalous pregnancies undergoing second-trimester genetic amniocentesis was followed up to delivery and analyzed as a nested case-control study. Cases of preterm birth (n = 52) were compared with 104 term control subjects. Amniotic fluid collected at amniocentesis was tested for TAT.
Results
TAT concentrations were significantly higher in women who delivered preterm (median 115.9 µg/L) than in those who did not (median 62.2 µg/L; P < .001). This difference persisted when 31 spontaneous preterm births and 21 indicated preterm births were analyzed separately. The odds ratios for preterm birth in the highest TAT quartile relative to the lowest quartile was 4.98 (95% confidence interval, 1.17–22.01; P = .007).
Conclusion
We found a difference in the pattern of intraamniotic thrombin generation between women destined to deliver at term and those who deliver preterm, regardless of the type of preterm birth.
Despite significant research efforts, the pathogenesis of preterm delivery remains unknown and the human burden of prematurity is growing. A pathogenic role for thrombosis was proposed by Arias et al in 1998. Specifically, thrombin appears to play a role in the physiologic pathways that link inflammation to uterine contractility, with increased thrombin generation acting as an autocrine/paracrine mediator for the onset of parturition, including preterm labor. Natural anticoagulant mechanisms limit the thrombin action by using antithrombin III to form stable stoichiometric complexes known as the thrombin-antithrombin (TAT) complexes. Because the direct measurement of thrombin is difficult, researchers have relied on indirect evidence of thrombin activation, such as markers of in vivo thrombin generation like TAT complexes. TAT complexes are demonstrable in the plasma and amniotic fluid of all pregnant women. Plasma TAT concentrations increase in pregnancy compared to the nonpregnant state, reflecting the state of excessive thrombin generation during pregnancy. The levels in the amniotic fluid are higher than in plasma, and increase from the second trimester toward term, with further increase in labor.
Studies on women with threatened preterm delivery in the third trimester have reported higher plasma and amniotic fluid TAT concentrations in women who deliver preterm than in women who deliver at term, and the higher the TAT levels, the shorter the latency period from presentation until delivery. Previous investigations of TAT complexes in pregnancy have also indicated that higher median plasma TAT concentrations in the second and third trimester precede the development of pregnancy complications such as preterm labor or preterm premature rupture of membranes (PPROM), suggesting that these complications may be preceded by decidual thrombin activation.
The relation between TAT levels in the amniotic fluid in asymptomatic women, early in pregnancy, and subsequent preterm labor or PPROM has not been well characterized. Thrombin generation, as expressed by TAT levels, is demonstrable at a significantly higher intensity in the amniotic fluid than in plasma, and amniotic fluid levels of TAT complexes are more likely to reflect pathologic alterations of the local environment than circulating levels, as previously described for certain cytokines. The primary objective of our study was to determine if amniotic fluid TAT concentrations measured in the second trimester correlate with subsequent preterm delivery.
Materials and Methods
The study was performed according to a protocol approved by our institutional Committee for the Protection of Human Subjects (HSC-MS-07-0109). The study cohort included women with singleton nonanomalous pregnancies undergoing second-trimester genetic amniocentesis between 15-26 weeks’ gestation. The subjects were prospectively enrolled, followed to delivery, and analyzed as a nested case-control study based on the outcome (timing of delivery). Subjects were excluded from the analysis if the amniocentesis results indicated abnormal fetal karyotype or confirmed suspected intraamniotic infection. Women who delivered at term served as controls for subject cases of preterm delivery. Controls were matched 2:1 for each case of preterm delivery based on the day of amniotic fluid collection to control for duration of specimen storage (±2 days). Demographic characteristics and pertinent maternal medical and obstetrical history were recorded at the time of enrollment and the circumstances of delivery were determined by review of the hospital computerized record verified by telephone interviews with the enrolled women and/or their primary obstetricians if necessary.
At the time of genetic amniocentesis, the first 2 mL of amniotic fluid, routinely discarded, were collected for study purpose and transported in a capped sterile tube to the laboratory where the sample was centrifuged for 10 minutes at 4°C. The supernatant was aliquoted and stored at −80°C until assay. Sensitive and specific quantitative enzyme-linked immunoassays for the detection of human TAT complexes (AssayMax human TAT complexes enzyme-linked immunosorbent assay kit; Gentaur, Kampenhout, Belgium) were carried out according to manufacturer’s recommendations by a technician blinded to the outcome. The calculated intraassay and interassay coefficients of variation for TAT were 6.3% and 8.2%, respectively. The assay sensitivity for TAT was <0.4 µg/L.
TAT complexes level was the main explanatory variable of interest and it was examined as a continuous and categorical variable (based on quartiles of TAT concentration). Differences in medians of continuous variables were tested using the Mann-Whitney test when 2 groups were compared and the Kruskal-Wallis analysis of variance test when >2 groups were compared. Differences between categorical variables were examined using the χ 2 test and odds ratios (ORs) were developed as a measure of association between dichotomous variables. The primary outcome was preterm delivery, spontaneous or indicated. Stratified analysis and the Mantel-Haenszel method were used to estimate the OR for preterm delivery adjusted for established risk factors for preterm delivery. The statistical software package used was STATA 10.0 (STATA Corp, College Station, TX). Women were considered to have had spontaneous preterm delivery if they experienced spontaneous onset of uterine contractions and delivered between 20-36 completed weeks’ gestation, or if they experienced PPROM and consequently delivered <36 completed weeks’ gestation. Indicated preterm delivery was defined as the iatrogenic initiation of delivery in cases of pregnancy complications such as preeclampsia, fetal demise, fetal growth restriction, or placental abruption, in the absence of spontaneous labor. Because the study endpoint was associated with gestational age at delivery, the accuracy of dating was essential. Gestational age was determined by known last menstrual period if consistent with fetal biometry at the time of amniocentesis, or by early sonogram if last menstrual period was unsure or there was >7 days difference between menstrual and ultrasound dates. It is known that menstrual dating may systematically overestimate gestational age by 1 week. For this reason, we selected as controls only women who delivered at ≥38 weeks’ gestation to ensure that control deliveries were indeed at term.
Results
Our final cohort comprised 550 pregnant women undergoing genetic amniocentesis between 15-26 weeks’ gestation. The median gestational age at amniocentesis was 17.7 weeks’ gestation (interquartile range [IQR], 3). Excluded from the analytic dataset after enrollment and amniocentesis were 24 cases (8 cases of trisomy 21, 2 cases of trisomy 18, 1 case of monosomy X, 1 case of 47, XXY, 1 case of deletion 9p24, 1 case of cytomegalovirus intraamniotic infection, 6 cases lost to follow-up, 1 case of incorrect data recording, 1 inadequate sample, and 1 case each of preterm delivery for placenta previa and uncontrolled diabetes mellitus). The enrollment period extended from June 2007 through March 2009.
In the entire cohort, the rate of preterm delivery was 9.5%, with mean gestational age at preterm delivery of 32.6 weeks’ gestation (range, 21–36). Of all preterm deliveries, 31 were spontaneous and 21 indicated (12 cases of preeclampsia, 5 of fetal demise, 3 cases of fetal growth restriction, and 1 placental abruption).
TAT complexes were identified in all 156 samples tested. The concentration values were not normally distributed and ranged from 1.6 µg/L in a woman who subsequently delivered at term, to 317.1 µg/L in a renal transplant recipient who developed severe preeclampsia, requiring delivery at 23 weeks’ gestation. Women who delivered preterm had a higher median amniotic fluid TAT complexes concentration (115.9 µg/L; IQR, 53.2–177.5) than those who delivered at term (62.2 µg/L; IQR, 38.9–98.8; P < .001). The median amniotic fluid TAT concentration did not differ significantly between women with spontaneous (100.6 µg/L; IQR, 49.9–159.8) and indicated (141.0 µg/L; IQR, 77.9–231.8; P = .20) preterm delivery. Similar trends were seen when spontaneous and indicated preterm deliveries were analyzed separately. In both subgroups, the median TAT concentration was significantly higher in women delivering preterm compared to their controls: 100.6 µg/L; IQR, 49.9–159.8 vs 61.8 µg/L; IQR, 40.4–101.1; P = .04 for spontaneous preterm delivery and 141.0 µg/L; IQR, 77.9–231.8 vs 63.2 µg/L; IQR, 38.8–94.8; P = .004 for indicated preterm delivery ( Figure ).
