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Feasibility of noninvasive prenatal testing for common fetal aneuploidies in an early gestational window
Clinica Chimica Acta 439 (2015) 24–28
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Feasibility of noninvasive prenatal testing for common fetal aneuploidies in an early gestational window Xiaolin Shi a, Zhitao Zhang a, David S. Cram b,⁎, Caixia Liu a,⁎⁎ a b
Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, Liaoning, China Berry Genomics, Beijing
a r t i c l e
i n f o
Article history: Received 29 August 2014 Accepted 29 September 2014 Available online 5 October 2014 Keywords: Early gestation Fetal aneuploidies Non-invasive prenatal testing Cell free fetal DNA fraction
a b s t r a c t Background: Noninvasive prenatal testing (NIPT) by massively parallel sequencing (MPS) of the circulating cell free fetal (cff) DNA during the second trimester of pregnancy is now a frontline test for detecting common fetal chromosomal abnormalities. However, the availability of an earlier test result in the first trimester would enable better clinical management of high-risk pregnancies. The aim of the study was to determine the feasibility of early gestational NIPT. Methods: Plasma DNA libraries were subjected to MPS and chromosomal read counts normalized to reference. Chromosomal aneuploidy was determined by z-scores (−3 b z b 3, normal range). The cff DNA fraction in 96 male pregnancies was calculated by the relative proportion of Y chromosomal reads. Results: NIPT results were obtained in the first (8–12 weeks) and second (15–18 weeks) trimester for 182 high-risk women. NIPT identified T21, T13 and 45,X in 3 pregnancies that were confirmed by karyotyping, but missed a T15 pregnancy that eventually miscarried. In the remaining 178 pregnancies, results for first and second trimester NIPT were normal. The median fetal fraction in the first trimester was 7.6 ± 4.18% and 15% of samples were identified with a cff fraction below 4%. Different trends of cff DNA fraction change were observed between the first and second trimester, with 59% of pregnancies showing an increase, 17% showing no change and 24% showing a decrease. Conclusions: Although NIPT was highly reliable and accurate at an earlier gestational age, clinical implementation should proceed with caution due to a small, but significant, number of pregnancies associated with a low cff DNA fraction. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Noninvasive prenatal diagnosis testing (NIPT) using massively parallel sequencing (MPS) of the circulating cell free maternal plasma DNA is now being used as a first tier test to assess the fetus for common chromosome abnormalities such as autosomal trisomies T21, T18 and T13 as well as sex chromosome aneuploidies [1,2]. In prospective clinical studies [3–6], the test sensitivities and specificities for T21, T18 and T13 are remarkably high, with less than 0.1% false positive and negative results. NIPT is available for both high and low risk pregnancies and is normally scheduled in the second trimester and occasionally, has been performed in the first trimester as early as 10 weeks gestation [2,7]. The cell free fetal (cff) DNA in the maternal plasma during pregnancy is believed to originate largely from the placental cells [8,9]. For clinical NIPT, the accuracy of the test is dependent on sufficient cff DNA fraction and most test formats are sensitive down to a threshold level ⁎ Co-corresponding author. ⁎⁎ Corresponding author. Tel.: +86 24 96615 43221. E-mail addresses: [email protected] (D.S. Cram), [email protected] (C. Liu).
http://dx.doi.org/10.1016/j.cca.2014.09.032 0009-8981/© 2014 Elsevier B.V. All rights reserved.
of approximately 4%. In the second trimester the average cff DNA fraction is 10–21% and rises approximately 1% each gestational week [10, 11]. High maternal weight [10,12,13], multiple gestations [14,15], increasing gestational age [10,12] and preeclampsia [11,16] are the most significant factors associated with a higher cff DNA fraction although secondary factors such as the presence of trisomy 21 [17], physical exercise [18], various fetal characteristics [13] and adverse pregnancy outcomes [11] have also been shown to modulate the levels of cff DNA in some patients. However, even with knowledge of these factors, it is still not possible to predict patients with a high or low cff DNA fraction suggesting that a number of other factors can regulate the final steady state of the fetal and maternal DNA fraction circulating in the plasma of each pregnant women [11]. The availability of NIPT at an earlier gestational timepoint would benefit women at high risk and alleviate the stress and anxiety of waiting for a result in the second trimester. To assess the feasibility of early gestational NIPT we compared the performance of our test in a cohort of 182 high risk pregnancies whereby sampling was performed between 8 and 12 week gestation in the first trimester and then repeated for each patient between 15 and 18 weeks in the second trimester. Here we report that early gestational NIPT is highly reliable and accurate even
X. Shi et al. / Clinica Chimica Acta 439 (2015) 24–28
at a generally lower cff DNA fraction compared to the second trimester. In addition, against current dogma, we show for the first time that the levels of cff DNA can fluctuate widely in each patient, with pregnancies showing either an increase, decrease or no appreciable change in cff DNA between the first and second trimester. 2. Methods 2.1. Study design The research study involved 209 high-risk pregnant women and was approved by the Institutional Review Board of Shengjing Hospital of China Medical University, Liaoning. Risk factors included either a history of miscarriage and/or advanced maternal age. For NIPT, patients consented to the collection of peripheral blood samples at two gestational age time points. The first sample was collected between 8 and 11+6 weeks in the first trimester and the second sample was collected between 15 and 18 weeks in the second trimester. In those pregnancies where NIPT results were positive, amniocentesis and karyotyping was conducted between 15 and 20 weeks. 2.2. Collection and processing of blood samples Peripheral blood samples (10 ml) were collected in Streck tubes and maternal plasma was separated within 48 h. Streck tubes were centrifuged at 1600 ×g for 10 min and the plasma layer was transferred to eppendorf tubes. Tubes were centrifuged at 16,000 ×g for 10 min and plasma aliquots carefully transferred to fresh eppendorf tubes. Purified plasma samples were stored at −20 °C. 2.3. Noninvasive prenatal testing Plasma DNA was prepared from each blood sample using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Library construction using barcoded adaptor primers, MPS on the Illumina 2000 platform and data analysis was performed according to previously published methods [3,19]. The fetal aneuploidy status for all 24 chromosomes was determined by z-scores (−3 b z b 3, normal range). For all pregnancies with a male fetus, calculation of the fetal DNA fraction was based on the percentage of Y chromosome reads, as previously described [20]. 3. Results 3.1. NIPT analysis of early and late gestation samples A total of 209 high-risk women consented to the study and presented for NIPT in the first trimester. NIPT identified nine aneuploidies comprising T21 (n = 4), T13 (n = 4) and 45,X (n = 1) (Table 1). Following early NIPT, three women with T21 fetuses and three women with T13 fetuses miscarried. Unfortunately, no miscarriage tissue was recovered and thus the initial NIPT results could not be confirmed. In the remaining 203 women with ongoing pregnancies, 21 women did not present for their second NIPT appointment. Therefore, a total of 182 patients
25
progressed to NIPT testing in the second trimester, including the three women with abnormal T21, T13 and 45,X NIPT results and the 179 women with normal NIPT results. Second trimester NIPT confirmed the three chromosomal abnormalities T21, T13 and 45,X detected by first trimester NIPT (Table 1). Follow-up amniocentesis and karyotyping also verified the three aneuploidies and the women elected to have pregnancy terminations. For the 179 pregnancies with normal first trimester NIPT results, 178 returned a normal result in the second trimester samples, indicating 100% sensitivity and specificity for T21, T13 and 45,X. However, in the one remaining pregnancy, the woman suffered a miscarriage prior to collection of blood for her second trimester NIPT. Karyotyping of the recovered miscarriage tissue by CNV sequencing [21] identified T15 (Fig. 1). Retrospective analysis of the NIPT results for this pregnancy revealed a z-score for Chr15 within the normal range (Table 1), indicating a false negative T15 by NIPT. 3.2. Analysis of cff DNA fraction in first and second trimester pregnancies In the 181 women who had repeat NIPT, 96 carried male fetuses (53%), allowing investigation of the trends in cff DNA fraction in the first and second trimesters. In the first trimester, the median ± 1SD% cff DNA was 7.6 ± 4.18% with a range of 2.56–21.03% (Fig. 2A). Within this gestational window of 51–84 days, there was a modest trend toward a higher cff DNA fraction with increasing gestational age (R2 = 0.109, p b 0.001). Of greater significance however, were 15 of 96 samples (15.6%) with a cff DNA of b4%, which is the sensitivity cut off for detection of T21 in our assay [3,19]. The samples with b4% cff DNA, however, were randomly distributed according to gestational age. In the second trimester, the median ± 1SD% cff DNA was 10.47 ± 4.70% (range 3.93– 31.45%) with 95 of 96 samples (99%) having a cff DNA fraction above 4% (Fig. 2B). There was only a slight trend toward a higher cff DNA fraction with increasing gestational age (R2 = 0.005), which was not significant (p = 0.48), presumably due to the clustering of samples collected in a narrow gestational window of 105–120 days. Overall, although there was a higher median cff DNA fraction in the second compared to the first trimester, the difference was not significant (p N 0.05). 3.3. Comparison of cff DNA fraction in first and second trimester pregnancies NIPT analysis of the 96 male pregnancies at the early and late gestational age also provided the opportunity to assess the change in cff DNA levels from the first to second trimester in the same pregnancies (Fig. 3). From the overall data, three trends were observed where the cff DNA fraction either increased by more than 1%, did not vary by more than 1% or decreased by more than 1%. From the 96 male pregnancies, 57 (59%) showed an increase cff DNA fraction, 16 (17%) showed no appreciable change in cff DNA fraction and 23 (24%) showed a decrease in cff DNA fraction. In the 23 pregnancies with a decline in cff DNA, the vast majority (78%) showed a N 3% drop in cff DNA fraction compared to the first trimester measurement. Interestingly, in all the 15 first trimester pregnancies with cff DNA
Table 1 First and second trimester NIPT abnormal results. Pregnancy
NIPT first trimester
NIPT1/NIPT2
Gestational age (days)
Chr z-scorea
Aneuploidy
Gestational age (days)
12SJ347/12SJ325 12SJ289/12SJ181 12SJ288/12SJ197 12SJ110
71 61 77 56
4.62 5.52 −3.35 2.74
T21 T13 45,X None
97 5.79 84 9.69 112 −7.03 Miscarriage after first NIPT sampling
a
NIPT second trimester
Normal chromosome copy number (−3 b z b 3).
Chr z-scorea
Karyotyping Aneuploidy T21 T13 45,X
T21 T13 45,X T15
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X. Shi et al. / Clinica Chimica Acta 439 (2015) 24–28
Fig. 1. Karyotyping of tissue from spontaneous miscarriage by CNV-Seq. Chromosome plots of log2 of mean CNV (Y-axis) versus each 20 kb sequencing read bin (X-axis) are shown for Chr15 (test) and Chr21 (control). The position of the blue line at log2[1.5], log2[0] and log2[0.5] represent 3, 2 and 1 copies of the chromosome. The miscarriage sample was identified with T15.
levels b 4%, there was a significant increase in cff DNA by the second trimester, with values well above the 4% threshold.
4. Discussion In this study we assessed the performance of NIPT in the last four weeks of the first trimester in a starting cohort of 209 high risk pregnancies, which represents a much earlier gestational window currently used in clinical practice. Based on the second trimester NIPT results and follow-up karyotyping of positive results, early gestation NIPT exhibited a similar accuracy and reliability to that previously reported for almost 10,000 pregnancies tested in the second trimester [3–6]. Three fetal trisomies were detected and confirmed by both second trimester NIPT and karyotyping. In the remaining pregnancies, NIPT results were negative when tested in the first and second trimester, suggesting a very high specificity. Although the study number is relatively small, the findings translate to a 100% sensitivity for detection of T21, T13 and 45,X. In addition, the six pregnancies that miscarried after the first NIPT was performed were either T21 or T13 fetus, lending further support to the high sensitivity of early NIPT for T21 and T13 detection. However, in one case, first trimester NIPT failed to detect a T15 that was later confirmed by karyotyping of the miscarriage tissue. This may have been due to a low cff DNA fraction, which was not measurable since the pregnancy involved a female fetus. Alternatively, T15 may have been missed because our NIPT assay has never been validated for T15 detection [3,19,22].
In the 8–12 week gestational window of the first trimester, the median cff DNA fraction for 96 male pregnancies was 7.6%. Comparatively for the same pregnancies, the median cff DNA fraction in the second trimester was 10.47%, a value that is consistent with other studies reporting an average cff DNA fraction of 10–21% in the second trimester [10,11]. Reviewing the data from the first trimester pregnancies, there was a relatively random distribution of cff DNA fractions across gestational age suggesting that there was a considerable degree of heterogeneity in this cohort of pregnant Chinese women of relatively similar weight and demographics. Of clinical significance were 15% of samples with cff DNA below 4% which is the cut-off for detection of T21 [3,19], and 3% of samples with cff DNA below 3% which is the cut-off for T18 and T13 detection [3,19]. Based on the assumption that the cff DNA fraction should be relatively similar in women carrying males or female pregnancies, the data suggest that our NIPT assay would be technically invalid for T21 in approximately 15% of samples and technically invalid for T18 and T13 in approximately 3% of samples collected in the first trimester between 8 and 12 week gestation. Prospective comparisons of cff DNA levels in both the first and second trimester for each of the 96 male pregnancies revealed different trends that have not been previously reported. Consistent with current dogma [10,11], there was a significant increase in cff DNA fraction for the majority of the pregnancies (59%). Unexpectedly, 17% of pregnancies showed no appreciable change in cff DNA fraction while the remaining 24% of pregnancies showed a significant decrease. In two of these 23 pregnancies, the cff DNA levels dropped 9.91% and 13.88% over 29 and 64 days, respectively. We
Fig. 2. Distribution of cff DNA levels with gestational age. The association between cff DNA fraction and gestational age was determined by the Pearson correlation co-efficient test.
X. Shi et al. / Clinica Chimica Acta 439 (2015) 24–28
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Fig. 3. Trends in cff DNA change between first and second trimester in individual pregnancies. Circled data points indicate a decrease in cff DNA fraction possibly due to a vanishing twin.
speculate that such a dramatic drop in cff DNA in these two pregnancies may have been attributable to a vanishing twin [23]. In the remaining 21 pregnancies with a decreased cff DNA fraction, we further speculate that a sharp gain in maternal weight from the first to second trimester relative to a steady increase in placental weight may be a possible causative factor. However, prospective measurement of maternal weight was not undertaken in this study and therefore further evidence to support this hypothesis is needed. Clearly, more carefully designed prospective studies on a larger cohort of pregnant women are required with multiple blood samplings, followed by collection of corresponding data on parameters such as maternal weight gain, placental growth, placental function and blood flow at the fetal–maternal interface. By this type of study, it may be possible to identify the key factors that modulate cff DNA fraction during early pregnancy [11]. In light of our findings that cff DNA fraction is highly variable and unpredictable in the first trimester together with the observation that approximately 15% of pregnancies have a low cff levels below 4%, clinical implementation of early NIPT will be challenging with the currently available sequencing technologies. One approach would be to initially offer early gestation NIPT to women with high-risk pregnancies and only report results where the cff DNA fraction is greater than 4%. In cases where the cff DNA fraction was determined to be less than 4%, the woman would be informed to reschedule a repeat NIPT in the second trimester to ensure a valid NIPT result. However, such an approach may ultimately prove clinically untenable if the repeat sampling rate exceeds 20%. A more plausible approach would be to improve the sensitivity of the test for low cff DNA fraction. Two strategies are emerging as promising solutions to detect levels of cff DNA below 4%. The first approach involves deeper sequencing of the plasma DNA to generate
more chromosomal reads and this has been shown to detect T21 at a level of 3% cff DNA fraction [24]. However, the downside is that deeper sequencing will significantly increase the overall cost of the test for patients. The second approach takes advantage of the overall difference in size distribution between the fetal and maternal populations of plasma DNA molecules [25], allowing size-based MPS method to determine fetal aneuploidy even at a low cff fraction [26]. The implementation of new strategies with a proven higher sensitivity at low cff DNA fraction will ultimately make early gestation NIPT feasible for the vast major of pregnancies. In conclusion, based on the successful performance of early gestation NIPT in this study for aneuploidy detection, but taking into consideration the issues associated with low cff DNA fraction, introduction of early gestation NIPT into clinical practice should proceed with caution. Advances in technology will no doubt increase the accuracy of the test to a level that will hopefully provide similar sensitivity and specificity as the current tests conducted in the second trimester NIPT. Once this is achieved, early gestation NIPT will become an attractive option for many couples to significantly reduce the waiting time for a diagnostic result and alleviate the anxiety and stress associated with a possible affected fetus. References [1] Tsui NB, Lo YM. Recent advances in the analysis of fetal nucleic acids in maternal plasma. Curr Opin Hematol 2012;19:462–8. [2] Bianchi DW, Wilkins-Haug L. Integration of noninvasive DNA testing for aneuploidy into prenatal care: what has happened since the rubber met the road? Clin Chem 2014;60:78–87. [3] Song Y, Liu C, Qi H, Zhang Y, Bian X, Liu J. Non invasive prenatal testing of fetal aneuploidies by massively parallel sequencing in a prospective Chinese population. Prenat Diagn 2013;33:700–6.
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[4] Bianchi DW, Parker RL, Wentworth J, et al. DNA sequencing versus standard prenatal aneuploidy screening. New Engl J Med 2014;370:799–808. [5] Lau TK, Cheung SW, Lo PS, et al. Non-invasive prenatal testing for fetal chromosomal abnormalities by low-coverage whole-genome sequencing of maternal plasma DNA: review of 1982 consecutive cases in a single center. Ultrasound Obstet Gynecol 2014;43:254–64. [6] Porreco RP, Garite TJ, Maurel K, et al. Noninvasive prenatal screening for fetal trisomies 21, 18, 13 and the common sex chromosome aneuploidies from maternal blood using massively parallel genomic sequencing of DNA. Am J Obstet Gynecol Mar 19 2014. http://dx.doi.org/10.1016/j.ajog.2014.03.042 [Epub ahead of print]. [7] Benn P, Cuckle H, Pergament E. Non-invasive prenatal testing for aneuploidy: current status and future prospects. Ultrasound Obstet Gynecol 2013;42:15–33. [8] Bianchi DW. Circulating fetal DNA: its origin and diagnostic potential—a review. Placenta 2004;25:S93–S101 [Suppl. A]. [9] Alberry M, Maddocks D, Jones M, et al. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenat Diagn 2007;27:415–8. [10] Canick JA, Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE. The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn 2013;33:667–76. [11] Taglauer ES, Wilkins-Haug L, Bianchi DW. Review: cell-free fetal DNA in the maternal circulation as an indication of placental health and disease. Placenta Feb 2014; 35:S64–8 [Suppl.]. [12] Wang E, Batey A, Struble C, et al. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma. Prenat Diagn 2013;33:662–6. [13] Ashoor G, Poon L, Syngelaki A, Mosimann B, Nicolaides KH. Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks' gestation: effect of maternal and fetal factors. Fetal Diagn Ther 2012;31:237–43. [14] Qu JZ, Leung TY, Jiang P, et al. Noninvasive prenatal determination of twin zygosity by maternal plasma DNA analysis. Clin Chem 2013;59:427–35.
[15] Leung TY, Qu JZ, Liao GJ, et al. Noninvasive twin zygosity assessment and aneuploidy detection by maternal plasma DNA sequencing. Prenat Diagn 2013;33:675–81. [16] Martin A, Krishna I, Martina B, Samuel A. Can the quantity of cell-free fetal DNA predict preeclampsia: a systematic review. Prenat Diagn 2014;34:685–91. [17] Rava RP, Srinivasan A, Sehnert AJ, Bianchi DW. Circulating fetal cell-free DNA fractions differ in autosomal aneuploidies and monosomy X. Clin Chem 2014;60: 243–50. [18] Schlütter JM, Hatt L, Bach C, Kirkegaard I, Kølvraa S, Uldbjerg N. The cell-free fetal DNA fraction in maternal blood decreases after physical activity. Prenat Diagn 2014;34:341–4. [19] Liang D, Lv W, Wang H, et al. Non-invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing. Prenat Diagn 2013;33:409–15. [20] Hudecova I, Sahota D, Heung MM, et al. Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies. PLoS One 2014; 9:e88484. [21] Liang D, Peng Y, Lv W, et al. Copy number variation sequencing for comprehensive diagnosis of chromosome disease syndromes. J Mol Diagn 2014;16:519–26. [22] Shaw SW, Hsiao CH, Chen CY, et al. Noninvasive prenatal testing for whole fetal chromosomal aneuploidies: a multicenter prospective cohort trial in Taiwan. Fetal Diagn Ther 2014;35:13–7. [23] Blickstein I, Perlman S. Single fetal death in twin gestations. J Perinat Med 2013;41: 65–9. [24] Benn P, Cuckle H. Theoretical performance of non-invasive prenatal testing for chromosome imbalances using counting of cell-free DNA fragments in maternal plasma. Prenat Diagn 2014;34:778–83. [25] Chan KC, Zhang J, Hui AB, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88–92. [26] Yu SC, Chan KC, Zheng YW, et al. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing. Proc Natl Acad Sci U S A 2014;111:8583–8.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Feasibility of noninvasive prenatal testing for common fetal aneuploidies in an early gestational window Xiaolin Shi a, Zhitao Zhang a, David S. Cram b,⁎, Caixia Liu a,⁎⁎ a b
Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, Liaoning, China Berry Genomics, Beijing
a r t i c l e
i n f o
Article history: Received 29 August 2014 Accepted 29 September 2014 Available online 5 October 2014 Keywords: Early gestation Fetal aneuploidies Non-invasive prenatal testing Cell free fetal DNA fraction
a b s t r a c t Background: Noninvasive prenatal testing (NIPT) by massively parallel sequencing (MPS) of the circulating cell free fetal (cff) DNA during the second trimester of pregnancy is now a frontline test for detecting common fetal chromosomal abnormalities. However, the availability of an earlier test result in the first trimester would enable better clinical management of high-risk pregnancies. The aim of the study was to determine the feasibility of early gestational NIPT. Methods: Plasma DNA libraries were subjected to MPS and chromosomal read counts normalized to reference. Chromosomal aneuploidy was determined by z-scores (−3 b z b 3, normal range). The cff DNA fraction in 96 male pregnancies was calculated by the relative proportion of Y chromosomal reads. Results: NIPT results were obtained in the first (8–12 weeks) and second (15–18 weeks) trimester for 182 high-risk women. NIPT identified T21, T13 and 45,X in 3 pregnancies that were confirmed by karyotyping, but missed a T15 pregnancy that eventually miscarried. In the remaining 178 pregnancies, results for first and second trimester NIPT were normal. The median fetal fraction in the first trimester was 7.6 ± 4.18% and 15% of samples were identified with a cff fraction below 4%. Different trends of cff DNA fraction change were observed between the first and second trimester, with 59% of pregnancies showing an increase, 17% showing no change and 24% showing a decrease. Conclusions: Although NIPT was highly reliable and accurate at an earlier gestational age, clinical implementation should proceed with caution due to a small, but significant, number of pregnancies associated with a low cff DNA fraction. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Noninvasive prenatal diagnosis testing (NIPT) using massively parallel sequencing (MPS) of the circulating cell free maternal plasma DNA is now being used as a first tier test to assess the fetus for common chromosome abnormalities such as autosomal trisomies T21, T18 and T13 as well as sex chromosome aneuploidies [1,2]. In prospective clinical studies [3–6], the test sensitivities and specificities for T21, T18 and T13 are remarkably high, with less than 0.1% false positive and negative results. NIPT is available for both high and low risk pregnancies and is normally scheduled in the second trimester and occasionally, has been performed in the first trimester as early as 10 weeks gestation [2,7]. The cell free fetal (cff) DNA in the maternal plasma during pregnancy is believed to originate largely from the placental cells [8,9]. For clinical NIPT, the accuracy of the test is dependent on sufficient cff DNA fraction and most test formats are sensitive down to a threshold level ⁎ Co-corresponding author. ⁎⁎ Corresponding author. Tel.: +86 24 96615 43221. E-mail addresses: [email protected] (D.S. Cram), [email protected] (C. Liu).
http://dx.doi.org/10.1016/j.cca.2014.09.032 0009-8981/© 2014 Elsevier B.V. All rights reserved.
of approximately 4%. In the second trimester the average cff DNA fraction is 10–21% and rises approximately 1% each gestational week [10, 11]. High maternal weight [10,12,13], multiple gestations [14,15], increasing gestational age [10,12] and preeclampsia [11,16] are the most significant factors associated with a higher cff DNA fraction although secondary factors such as the presence of trisomy 21 [17], physical exercise [18], various fetal characteristics [13] and adverse pregnancy outcomes [11] have also been shown to modulate the levels of cff DNA in some patients. However, even with knowledge of these factors, it is still not possible to predict patients with a high or low cff DNA fraction suggesting that a number of other factors can regulate the final steady state of the fetal and maternal DNA fraction circulating in the plasma of each pregnant women [11]. The availability of NIPT at an earlier gestational timepoint would benefit women at high risk and alleviate the stress and anxiety of waiting for a result in the second trimester. To assess the feasibility of early gestational NIPT we compared the performance of our test in a cohort of 182 high risk pregnancies whereby sampling was performed between 8 and 12 week gestation in the first trimester and then repeated for each patient between 15 and 18 weeks in the second trimester. Here we report that early gestational NIPT is highly reliable and accurate even
X. Shi et al. / Clinica Chimica Acta 439 (2015) 24–28
at a generally lower cff DNA fraction compared to the second trimester. In addition, against current dogma, we show for the first time that the levels of cff DNA can fluctuate widely in each patient, with pregnancies showing either an increase, decrease or no appreciable change in cff DNA between the first and second trimester. 2. Methods 2.1. Study design The research study involved 209 high-risk pregnant women and was approved by the Institutional Review Board of Shengjing Hospital of China Medical University, Liaoning. Risk factors included either a history of miscarriage and/or advanced maternal age. For NIPT, patients consented to the collection of peripheral blood samples at two gestational age time points. The first sample was collected between 8 and 11+6 weeks in the first trimester and the second sample was collected between 15 and 18 weeks in the second trimester. In those pregnancies where NIPT results were positive, amniocentesis and karyotyping was conducted between 15 and 20 weeks. 2.2. Collection and processing of blood samples Peripheral blood samples (10 ml) were collected in Streck tubes and maternal plasma was separated within 48 h. Streck tubes were centrifuged at 1600 ×g for 10 min and the plasma layer was transferred to eppendorf tubes. Tubes were centrifuged at 16,000 ×g for 10 min and plasma aliquots carefully transferred to fresh eppendorf tubes. Purified plasma samples were stored at −20 °C. 2.3. Noninvasive prenatal testing Plasma DNA was prepared from each blood sample using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Library construction using barcoded adaptor primers, MPS on the Illumina 2000 platform and data analysis was performed according to previously published methods [3,19]. The fetal aneuploidy status for all 24 chromosomes was determined by z-scores (−3 b z b 3, normal range). For all pregnancies with a male fetus, calculation of the fetal DNA fraction was based on the percentage of Y chromosome reads, as previously described [20]. 3. Results 3.1. NIPT analysis of early and late gestation samples A total of 209 high-risk women consented to the study and presented for NIPT in the first trimester. NIPT identified nine aneuploidies comprising T21 (n = 4), T13 (n = 4) and 45,X (n = 1) (Table 1). Following early NIPT, three women with T21 fetuses and three women with T13 fetuses miscarried. Unfortunately, no miscarriage tissue was recovered and thus the initial NIPT results could not be confirmed. In the remaining 203 women with ongoing pregnancies, 21 women did not present for their second NIPT appointment. Therefore, a total of 182 patients
25
progressed to NIPT testing in the second trimester, including the three women with abnormal T21, T13 and 45,X NIPT results and the 179 women with normal NIPT results. Second trimester NIPT confirmed the three chromosomal abnormalities T21, T13 and 45,X detected by first trimester NIPT (Table 1). Follow-up amniocentesis and karyotyping also verified the three aneuploidies and the women elected to have pregnancy terminations. For the 179 pregnancies with normal first trimester NIPT results, 178 returned a normal result in the second trimester samples, indicating 100% sensitivity and specificity for T21, T13 and 45,X. However, in the one remaining pregnancy, the woman suffered a miscarriage prior to collection of blood for her second trimester NIPT. Karyotyping of the recovered miscarriage tissue by CNV sequencing [21] identified T15 (Fig. 1). Retrospective analysis of the NIPT results for this pregnancy revealed a z-score for Chr15 within the normal range (Table 1), indicating a false negative T15 by NIPT. 3.2. Analysis of cff DNA fraction in first and second trimester pregnancies In the 181 women who had repeat NIPT, 96 carried male fetuses (53%), allowing investigation of the trends in cff DNA fraction in the first and second trimesters. In the first trimester, the median ± 1SD% cff DNA was 7.6 ± 4.18% with a range of 2.56–21.03% (Fig. 2A). Within this gestational window of 51–84 days, there was a modest trend toward a higher cff DNA fraction with increasing gestational age (R2 = 0.109, p b 0.001). Of greater significance however, were 15 of 96 samples (15.6%) with a cff DNA of b4%, which is the sensitivity cut off for detection of T21 in our assay [3,19]. The samples with b4% cff DNA, however, were randomly distributed according to gestational age. In the second trimester, the median ± 1SD% cff DNA was 10.47 ± 4.70% (range 3.93– 31.45%) with 95 of 96 samples (99%) having a cff DNA fraction above 4% (Fig. 2B). There was only a slight trend toward a higher cff DNA fraction with increasing gestational age (R2 = 0.005), which was not significant (p = 0.48), presumably due to the clustering of samples collected in a narrow gestational window of 105–120 days. Overall, although there was a higher median cff DNA fraction in the second compared to the first trimester, the difference was not significant (p N 0.05). 3.3. Comparison of cff DNA fraction in first and second trimester pregnancies NIPT analysis of the 96 male pregnancies at the early and late gestational age also provided the opportunity to assess the change in cff DNA levels from the first to second trimester in the same pregnancies (Fig. 3). From the overall data, three trends were observed where the cff DNA fraction either increased by more than 1%, did not vary by more than 1% or decreased by more than 1%. From the 96 male pregnancies, 57 (59%) showed an increase cff DNA fraction, 16 (17%) showed no appreciable change in cff DNA fraction and 23 (24%) showed a decrease in cff DNA fraction. In the 23 pregnancies with a decline in cff DNA, the vast majority (78%) showed a N 3% drop in cff DNA fraction compared to the first trimester measurement. Interestingly, in all the 15 first trimester pregnancies with cff DNA
Table 1 First and second trimester NIPT abnormal results. Pregnancy
NIPT first trimester
NIPT1/NIPT2
Gestational age (days)
Chr z-scorea
Aneuploidy
Gestational age (days)
12SJ347/12SJ325 12SJ289/12SJ181 12SJ288/12SJ197 12SJ110
71 61 77 56
4.62 5.52 −3.35 2.74
T21 T13 45,X None
97 5.79 84 9.69 112 −7.03 Miscarriage after first NIPT sampling
a
NIPT second trimester
Normal chromosome copy number (−3 b z b 3).
Chr z-scorea
Karyotyping Aneuploidy T21 T13 45,X
T21 T13 45,X T15
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Fig. 1. Karyotyping of tissue from spontaneous miscarriage by CNV-Seq. Chromosome plots of log2 of mean CNV (Y-axis) versus each 20 kb sequencing read bin (X-axis) are shown for Chr15 (test) and Chr21 (control). The position of the blue line at log2[1.5], log2[0] and log2[0.5] represent 3, 2 and 1 copies of the chromosome. The miscarriage sample was identified with T15.
levels b 4%, there was a significant increase in cff DNA by the second trimester, with values well above the 4% threshold.
4. Discussion In this study we assessed the performance of NIPT in the last four weeks of the first trimester in a starting cohort of 209 high risk pregnancies, which represents a much earlier gestational window currently used in clinical practice. Based on the second trimester NIPT results and follow-up karyotyping of positive results, early gestation NIPT exhibited a similar accuracy and reliability to that previously reported for almost 10,000 pregnancies tested in the second trimester [3–6]. Three fetal trisomies were detected and confirmed by both second trimester NIPT and karyotyping. In the remaining pregnancies, NIPT results were negative when tested in the first and second trimester, suggesting a very high specificity. Although the study number is relatively small, the findings translate to a 100% sensitivity for detection of T21, T13 and 45,X. In addition, the six pregnancies that miscarried after the first NIPT was performed were either T21 or T13 fetus, lending further support to the high sensitivity of early NIPT for T21 and T13 detection. However, in one case, first trimester NIPT failed to detect a T15 that was later confirmed by karyotyping of the miscarriage tissue. This may have been due to a low cff DNA fraction, which was not measurable since the pregnancy involved a female fetus. Alternatively, T15 may have been missed because our NIPT assay has never been validated for T15 detection [3,19,22].
In the 8–12 week gestational window of the first trimester, the median cff DNA fraction for 96 male pregnancies was 7.6%. Comparatively for the same pregnancies, the median cff DNA fraction in the second trimester was 10.47%, a value that is consistent with other studies reporting an average cff DNA fraction of 10–21% in the second trimester [10,11]. Reviewing the data from the first trimester pregnancies, there was a relatively random distribution of cff DNA fractions across gestational age suggesting that there was a considerable degree of heterogeneity in this cohort of pregnant Chinese women of relatively similar weight and demographics. Of clinical significance were 15% of samples with cff DNA below 4% which is the cut-off for detection of T21 [3,19], and 3% of samples with cff DNA below 3% which is the cut-off for T18 and T13 detection [3,19]. Based on the assumption that the cff DNA fraction should be relatively similar in women carrying males or female pregnancies, the data suggest that our NIPT assay would be technically invalid for T21 in approximately 15% of samples and technically invalid for T18 and T13 in approximately 3% of samples collected in the first trimester between 8 and 12 week gestation. Prospective comparisons of cff DNA levels in both the first and second trimester for each of the 96 male pregnancies revealed different trends that have not been previously reported. Consistent with current dogma [10,11], there was a significant increase in cff DNA fraction for the majority of the pregnancies (59%). Unexpectedly, 17% of pregnancies showed no appreciable change in cff DNA fraction while the remaining 24% of pregnancies showed a significant decrease. In two of these 23 pregnancies, the cff DNA levels dropped 9.91% and 13.88% over 29 and 64 days, respectively. We
Fig. 2. Distribution of cff DNA levels with gestational age. The association between cff DNA fraction and gestational age was determined by the Pearson correlation co-efficient test.
X. Shi et al. / Clinica Chimica Acta 439 (2015) 24–28
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Fig. 3. Trends in cff DNA change between first and second trimester in individual pregnancies. Circled data points indicate a decrease in cff DNA fraction possibly due to a vanishing twin.
speculate that such a dramatic drop in cff DNA in these two pregnancies may have been attributable to a vanishing twin [23]. In the remaining 21 pregnancies with a decreased cff DNA fraction, we further speculate that a sharp gain in maternal weight from the first to second trimester relative to a steady increase in placental weight may be a possible causative factor. However, prospective measurement of maternal weight was not undertaken in this study and therefore further evidence to support this hypothesis is needed. Clearly, more carefully designed prospective studies on a larger cohort of pregnant women are required with multiple blood samplings, followed by collection of corresponding data on parameters such as maternal weight gain, placental growth, placental function and blood flow at the fetal–maternal interface. By this type of study, it may be possible to identify the key factors that modulate cff DNA fraction during early pregnancy [11]. In light of our findings that cff DNA fraction is highly variable and unpredictable in the first trimester together with the observation that approximately 15% of pregnancies have a low cff levels below 4%, clinical implementation of early NIPT will be challenging with the currently available sequencing technologies. One approach would be to initially offer early gestation NIPT to women with high-risk pregnancies and only report results where the cff DNA fraction is greater than 4%. In cases where the cff DNA fraction was determined to be less than 4%, the woman would be informed to reschedule a repeat NIPT in the second trimester to ensure a valid NIPT result. However, such an approach may ultimately prove clinically untenable if the repeat sampling rate exceeds 20%. A more plausible approach would be to improve the sensitivity of the test for low cff DNA fraction. Two strategies are emerging as promising solutions to detect levels of cff DNA below 4%. The first approach involves deeper sequencing of the plasma DNA to generate
more chromosomal reads and this has been shown to detect T21 at a level of 3% cff DNA fraction [24]. However, the downside is that deeper sequencing will significantly increase the overall cost of the test for patients. The second approach takes advantage of the overall difference in size distribution between the fetal and maternal populations of plasma DNA molecules [25], allowing size-based MPS method to determine fetal aneuploidy even at a low cff fraction [26]. The implementation of new strategies with a proven higher sensitivity at low cff DNA fraction will ultimately make early gestation NIPT feasible for the vast major of pregnancies. In conclusion, based on the successful performance of early gestation NIPT in this study for aneuploidy detection, but taking into consideration the issues associated with low cff DNA fraction, introduction of early gestation NIPT into clinical practice should proceed with caution. Advances in technology will no doubt increase the accuracy of the test to a level that will hopefully provide similar sensitivity and specificity as the current tests conducted in the second trimester NIPT. Once this is achieved, early gestation NIPT will become an attractive option for many couples to significantly reduce the waiting time for a diagnostic result and alleviate the anxiety and stress associated with a possible affected fetus. References [1] Tsui NB, Lo YM. Recent advances in the analysis of fetal nucleic acids in maternal plasma. Curr Opin Hematol 2012;19:462–8. [2] Bianchi DW, Wilkins-Haug L. Integration of noninvasive DNA testing for aneuploidy into prenatal care: what has happened since the rubber met the road? Clin Chem 2014;60:78–87. [3] Song Y, Liu C, Qi H, Zhang Y, Bian X, Liu J. Non invasive prenatal testing of fetal aneuploidies by massively parallel sequencing in a prospective Chinese population. Prenat Diagn 2013;33:700–6.
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