Our objective was to examine the relationship between cell-free DNA levels and serum markers in both first and second trimesters of pregnancy. Our analysis demonstrates that there is a marginal, yet statistically significant, correlation between first-trimester serum PAPP-A and first-trimester cell-free and total DNA levels. Both are markers of placental function, because PAPP-A is a glycoprotein of placental origin, and cell-free DNA in maternal serum derives predominantly from apoptosis of placental cells.
Concerning the biological plausibility for the correlation between PAPP-A and cell-free DNA, PAPP-A is a protease for insulin growth factor–binding protein 4. Lower levels of PAPP-A are associated with higher levels of insulin growth factor–binding protein 4, and thus, less free insulin growth factor. Insulin growth factor regulates fetal growth by stimulating cell proliferation and differentiation and plays a role in trophoblast invasion of the decidua.20,21 Cell-free DNA is thought to be mainly released from the placenta and is a result of trophoblastic apoptosis.5,22 If there is abnormal trophoblast invasion in early pregnancy and decreased subsequent cell proliferation, this could result in poor placental development and an increase in placental apoptosis, which would then result in high cell-free DNA levels with correspondingly low PAPP-A levels. Further studies are needed to test this hypothesis.
This study shows an early marginal correlation in the first trimester between PAPP-A and cell-free fetal DNA. In the second trimester, total and fetal DNA are not correlated with any of the serum analytes, even the analytes specifically of placental origin. This is surprising given that the source of fetal DNA measured in the maternal circulation is hypothesized to predominantly originate from the placenta. These results raise the question of extraplacental sources of fetal DNA and suggest that such sources may differ between trimesters. This possibility warrants further investigation as to the source of cell-free DNA throughout pregnancy.
Prior reports in the literature demonstrate that a low PAPP-A value and a high cell-free fetal DNA measurement are both associated with adverse pregnancy outcomes. Results from the First and Second Trimester Evaluation of Risk (FASTER) trial showed that a PAPP-A measurement of less than the fifth percentile was associated with spontaneous pregnancy loss at less than 24 weeks of gestation, preeclampsia, low birth weight, gestational hypertension, preterm birth, stillbirth, preterm premature rupture of membranes, and placental abruption.20 Low PAPP-A and high DNA levels both suggest placental abnormalities. Future work is needed to determine whether this correlation has clinical significance. Although abnormal serum markers are indicators of poor placental health and are associated with adverse pregnancy outcomes, there is no currently accepted practice for managing pregnancies in women with abnormal serum markers. Because cell-free fetal DNA can be detected in the first trimester, it may have use, in conjunction with other analytes, as an early marker of adverse obstetric outcomes.23
Markers in serum screening have been chosen based on their ability to provide independent information. Low correlation between serum markers is required to improve Down syndrome detection rates.24,25 Addition of fetal DNA as a marker of Down syndrome may improve screening performance, especially in the second trimester when all correlations are low. On the other hand, recent strategies have emerged in screening to take advantage of high correlations of marker levels. High correlation of a marker across trimesters can be used to improve prenatal screening for Down syndrome by calculating ratios of the levels of the same serum markers measured in the first and the second trimester (cross-trimester ratios).26 Use of cross-trimester ratios was also found to improve the performance of the integrated screening test and lower the false-positive rate.26 We found a statistically significant correlation between cell-free fetal DNA in the first and second trimesters (P=.015). Future work studying cell-free DNA levels across trimesters can be done to determine whether this could improve screening performance. However, the use of cell-free fetal DNA can ultimately only be realized with a gender-independent marker. In this study, a Y chromosome sequence was used a marker of fetal DNA, meaning that only half of pregnancies can be analyzed. Although these data show the feasibility of using cell-free DNA as a biomarker, a gender-independent marker is essential for widespread clinical implementation.
The correlation of second-trimester serum markers and fetal DNA has been examined in a prior study.10 Our results are in agreement that the correlation of markers is low. The correlation (r) of alpha-fetoprotein, unconjugated estriol, or β-hCG with fetal DNA ranged from 0.1 to 0.2 in both studies. The results for inhibin were more variable: 0.3 in the prior study but −0.09 presently. Nevertheless, either of these correlations is low enough to suggest a benefit from addition of fetal DNA levels to second-trimester screening. Indeed, Farina et al10 calculated that fetal DNA measurement could add 5% detection to second-trimester Down syndrome screening over routine quad markers alone.
A limitation of the study is that pregnancy outcomes were not determined other than to confirm that there was a birth of a liveborn singleton male. However, the objective of the study was to compare serum analytes with cell-free DNA levels toward potential future improvements to serum screening and not to assess for pregnancy outcome. In addition, as a result of the small sample, it is possible that marginal correlations that were reported (ie, correlation of cell-free fetal DNA and PAPP-A) or those that were undiscovered would be further delineated from a larger sample size. Nevertheless, there are a number of strengths in our study. We used the same patients for the first- and second-trimester data, 98% of patients were nonsmokers, and in vitro fertilization patients were excluded. Prior data has shown that smoking in pregnancy increases levels of total cell-free DNA threefold.27 In addition, although one study has shown that in vitro fertilization does not affect levels of cell-free fetal DNA,28 these pregnancies were excluded because some studies suggest that serum analytes in pregnancies conceived with assisted reproductive technologies may be abnormal.29
Our observation that PAPP-A and cell-free DNA are correlated in the first trimester reinforces the idea of cell-free DNA as a marker of placental development. Further studies should be done to determine whether pregnancies with abnormal placentas show an increase in trafficking of total and fetal DNA and whether this increase correlates with other placental secretory products. If patients with abnormally low PAPP-A and high cell-free DNA levels have poor pregnancy outcomes, these combined markers could potentially identify pregnancies in the first trimester that warrant increased surveillance.
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