Fetomaternal hemorrhage (FMH) during pregnancy or subsequent isoimmunization, caused by a breach of the placental barrier, could lead to an adverse perinatal outcome (1)(2). However, concerns have been raised regarding the accuracy of the standard Kleihauer–Betke test to quantify FMH (3). In a previously published study, we explored the possibility of using cell-free fetal DNA (fDNA) in maternal plasma as a novel marker of FMH after surgical and medical elective first-trimester termination of pregnancy (TOP) (4).
In our earlier study, pre- and posttermination blood samples were drawn at various time points relative to TOP. To adjust for this confounder, we retrospectively applied a mathematical model to the analysis of the data. Projected pretermination fDNA values were calculated based on the anticipated linear increase of fDNA during pregnancy. Adjusted posttermination fDNA concentrations were calculated based on the reported 16-min clearance rate of circulating fDNA after delivery (5). Adjusted posttermination fDNA concentrations were then compared with the projected pretermination concentrations.
In the present study, we specifically enrolled only pregnant women in the first trimester undergoing elective surgical TOP. In addition, the pre- and posttermination blood samples were drawn on the same day with a controlled time interval between TOP and the posttermination blood collection. The concentrations of circulating fDNA were then analyzed along with pertinent clinical information to better understand the dynamics of circulating fDNA after TOP.
This study was approved by the Institutional Review Boards at Tufts-New England Medical Center and Boston University School of Medicine. Pregnant women in the first trimester of pregnancy undergoing elective surgical TOP at Boston Medical Center were enrolled. Gestational ages were ascertained by ultrasonography and were expressed as postmenstrual days. Paired blood samples were obtained before and immediately after the termination procedure. The blood samples were centrifuged at 800g, after which plasma was isolated and frozen at −80 °C until analysis.
The patients received intravenous sedation with fentanyl and midazolam, or local anesthesia. No patients required bolus intravenous fluid. The products of conception (POC) were evacuated by use of manual vacuum aspiration, followed by sharp curettage at the discretion of the operator. The times of suction initiation and blood drawing were recorded. Products of conception were frozen in saline for fetal gender identification.
Plasma (900 μL) was centrifuged at 11 500g for 10 min to remove residual cells. We extracted DNA from 800 μL of the supernatant by use of the QIAamp Blood Kit (Qiagen Inc.) with the Blood and Body Fluid Spin Protocol described by the manufacturer. The extracted DNA was eluted into a final volume of 50 μL. We processed 500 μL of the normal saline solution that contained DNA diffused from the POC in the same fashion and used 400 μL of the supernatant for DNA extraction.
We measured fDNA in maternal plasma and the POC solution by real-time PCR amplification using a Perkin-Elmer Applied Biosystems 7700 Sequence Detector (Applied Biosystems). The DYS1 sequence on the Y chromosome was used to detect and quantify male DNA as described previously, with fDNA concentrations expressed in genome-equivalents (GE)/mL of plasma (6). For all samples, the β-globin gene sequence was amplified to estimate the total amount of genomic DNA in the sample.
Descriptive statistics, including medians with 25th and 75th percentile ranges, were generated for all continuous variables, and frequencies were generated for discrete variables. The Wilcoxon signed-rank test was used to assess the difference in median fDNA concentrations between pre- and posttermination samples. The women were classified as having either increased or decreased fDNA concentrations after TOP. Appropriate statistical tests were used to assess differences in continuous and discrete characteristics between women whose posttermination fDNA concentrations were increased and those whose concentrations were decreased as follows: the Mann–Whitney–Wilcoxon test was used to assess the differences in median pretermination fDNA concentrations, median time interval between TOP and post-TOP blood draw, and median gestational age between the two groups; the χ2 test was used to estimate the difference between groups in the percentage of women who received sharp uterine curettage; and the Fisher exact test was used to assess the difference in the percentages of the women in the two groups who had vaginal bleeding before the procedure. All analyses were performed with SAS/STAT, Ver. 8.2 (SAS Institute, Inc.).
Sixty-three patients were enrolled in the study. The median (25th, 75th percentiles) gestational age when TOP was performed was 66 (57.5, 73.5) days. The β-globin gene sequence was amplified in every plasma and POC sample analyzed. Male DNA was amplified in 31 of 63 POC samples, implying the presence of a male fetus. Fetal gender detection from maternal plasma was 100% accurate when compared with PCR analysis of the POC.
Among pregnancies with a male fetus, median (25th, 75th percentiles) pre- and posttermination fDNA concentrations were 12.3 (10.4, 19.8) and 14.9 (7.9, 22.4) GE/mL, respectively, giving a mean increase of 2.6 GE/mL. This difference, however, was not statistically significant (P = 0.98). In 14 of 31 (45%) patients, increased fDNA was observed after the pregnancy termination procedure. The gestational age was significantly more advanced in these patients (P = 0.04), as shown in Fig. 1⇓ .
There was no significant difference in the median concentration of pretermination fDNA between those patients with increased or decreased concentrations of posttermination fDNA (P = 0.34; Table 1⇓ ). Among the patients who carried a male conceptus, 17 of 31 (55%) received sharp curettage and 4 of 31 (13%) had vaginal bleeding before the procedure. The median (25th, 75th percentiles) interval between the procedure and the blood draw was 23 (20, 33) min. As summarized in Table 1⇓ , the frequencies of sharp curettage, vaginal bleeding, and the time interval between procedure and blood draw were not significantly different between women who had increased posttermination fDNA and those with decreased concentrations (P = 0.34, 1.00, and 0.89, respectively).
The data from the present study support our previous report that plasma fDNA is increased in some patients after elective first-trimester TOP (4). After controlling for potential confounding variables, approximately one half of the first-trimester pregnant patients had increased plasma fDNA after TOP. These women were more advanced in gestation than those in whom fDNA concentrations were decreased. This correlates with the development of the placental vasculature and thus suggests the contribution of DNA from fetal blood cells to the pool of circulating fDNA in maternal plasma after TOP.
Overall, the median concentration of post-TOP fDNA among all patients was not significantly increased over the median pre-TOP fDNA concentration, but our data show an increase in fDNA after TOP only after 8 weeks of gestation. This increase occurred in 11 of 17 patients when pregnancy was later than 9.5 weeks of gestation (Fig. 1⇓ ). The increase in fDNA at later gestations could be attributable to either an increase in placental size/mass or disruption of the fetomaternal circulation. Using Doppler studies, Jauniaux et al. (7) showed that placental blood flow is not established until 8–9 weeks of gestation. Thus, we would not necessarily expect to find FMH after TOP earlier than this gestational age. In support of this, Leong et al. (8) found no fetal cells in the blood samples of women after surgical TOP at <6 weeks menstrual age. We therefore suggest that the source of this DNA may be fetal hematopoietic cells within newly established placental blood vessels that are disrupted as a result of the TOP procedure and that the increased fDNA concentrations may indicate excessive FMH after 9 weeks of gestation.
Plasma fDNA concentrations in many patients, however, unexpectedly decreased after the TOP procedure. Because no intravenous fluid bolus was given to any patient before the procedure, a dilution effect is unlikely. This decrease might be explained instead by the physiologic differences in how fDNA enters the maternal circulation. It is generally believed that in pregnant women the source of cell-free fetal nucleic acids is placentally derived apoptotic cells (9). Some fDNA sequences are detectable in membrane-bound apoptotic vesicles (10). This particle-associated form is believed to protect fetal nucleic acids from degradation by nucleases in maternal blood (11). After elective TOP, fDNA is liberated directly into the maternal circulation from the sudden disruption of the fetomaternal interface; thus it may not be protected in apoptotic bodies. We therefore suggest that unprotected posttermination fDNA sequences are vulnerable to destruction by maternal nucleases, leading to the rapid decrease in these sequences after elective termination in some patients.
Future study should define the association between post-TOP cell-free fDNA concentrations, measured by real-time PCR, and cellular trafficking, determined by the traditional Kleihauer–Betke test, in a large cohort of patients at various gestational ages. In addition, the correlation between the alteration in fDNA concentrations and triggering of the maternal immune response remains to be elucidated. Current measurement of fDNA is based on Y-chromosome-specific sequence detection, which is not applicable to women who carry a female fetus. Therefore, the continued development of fetal gender-independent markers, such as fetal/placental specific mRNA, in the maternal circulation is essential (12). Comparison of fetal globin gene expression with placentally derived gene expression may allow us to definitively determine whether the increased fetal nucleic acids seen after 9 weeks of gestation in maternal plasma are from a placental or hematopoietic source (13).
Dr. Wataganara’s maternal-fetal medicine fellowship is supported in part by Anandamahidol Foundation, Thailand. We thank Olivera Vragovic for help in organizing the study and enrolling the patients, and Dr. Dittakarn Boriboonhirunsarn for insightful comments on the manuscript.
- © 2005 The American Association for Clinical Chemistry