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Technical Briefs |
Departments of1 Chemical Pathology and2 Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR;3 Department of Histology, Medical Embryology, Obstetrics and Gynecology, University of Bologna, Policlinico S. Orsola Malpighi, Bologna, Italy;
aaddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Room 38023, 1/F Clinical Sciences Building, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong Special Administrative Region, China; fax 852-2194-6171, e-mail loym{at}cuhk.edu.hk
Abstract
Background: Fetal RNA of placental origin has been detected in the plasma of pregnant women, but the timing of the first appearance and the detailed kinetics of postdelivery clearance of such circulating RNA have not been studied.
Methods: To address the timing of the first appearance of circulating placental RNA, we collected serial maternal blood samples from 47 women who had conceived by assisted reproductive procedures. To address the postdelivery clearance kinetics, we collected serial postdelivery blood samples from 6 pregnant women who had delivered by cesarean section. Placenta-derived transcripts were sought by real-time quantitative reverse transcription-PCR.
Results: The earliest gestational age at which human placental lactogen and human chorionic gonadotropin ß-subunit mRNAs were detectable in a proportion of the pregnant women was the 4th week of gestation. The postdelivery study indicated that the median apparent half-life for the clearance of human placental lactogen mRNA was 14 min.
Conclusions: Placenta-derived mRNA can be found in maternal plasma from very early on in gestation, suggesting a possible role for early noninvasive prenatal diagnosis or monitoring. The rapid kinetics of circulating placental mRNA suggest that its plasma concentrations may be used to monitor recent physiologic or pathologic events.
Detection of circulating fetal nucleic acids in maternal plasma has been a promising means for the development of noninvasive strategies for prenatal assessment and monitoring (1). In particular, mRNA molecules of placentally expressed genes are detectable in maternal plasma (2)(3), and they offer the opportunity for noninvasive placental gene expression profiling (4). The presence of such placenta-derived mRNA in the maternal circulation is pregnancy-specific (2)(4). Being free from interference by similar maternally expressed genes, circulating placental mRNA analysis offers a fetal gender- and polymorphism-independent means for noninvasive prenatal monitoring for conditions such as preeclampsia (3)(5) and fetal aneuploidy(6). However, the timing of first appearance and the detailed kinetics of postdelivery clearance of the placenta-derived circulating mRNA have not been previously addressed.
All samples in this study were collected with informed consent and institutional ethics approvals. To investigate the earliest point in gestation at which placental mRNA could be detected in maternal plasma, serial maternal blood was collected at weekly intervals from women who had conceived by assisted reproductive procedures (including in vitro fertilization, embryo transfer, and intrauterine insemination) at the Prince of Wales Hospital, Hong Kong. To study the clearance profile of placental mRNA, we collected postdelivery blood samples from women undergoing elective cesarean section at the Department of Obstetrics and Gynecology, University of Bologna, Italy. One sample was collected before the scheduled delivery, followed by a sample collected within 15 to 25 min after cesarean section, and 3 additional samples collected at 30-min intervals thereafter. Maternal blood samples were collected in EDTA tubes. Plasma was harvested (2), and 4 mL of Trizol LS reagent (Invitrogen) was mixed with 3.2 mL of plasma before storage at –80 °C. Plasma samples collected from Italy were sent on dry ice to Hong Kong for analysis. Plasma RNA was extracted as described previously (2). The authors of a previously reported microarray study reported that human placental lactogen (hPL) is highly expressed in both first- and third-trimester placental tissues, whereas human chorionic gonadotropin ß-subunit (ßhCG) is produced mainly in first-trimester placentas (4). Therefore, using real-time quantitative reverse transcription-PCR assays, as described previously (2), we tested for the presence of both transcripts in the maternal plasma samples collected during early pregnancy but only for hPL mRNA in the postdelivery samples. Both assays were sensitive to the detection of 10 copies of the mRNA transcript per reaction, corresponding to 
30 copies/mL of plasma.
Forty-seven women who had conceived by assisted reproduction consented to participate in the study. As determined by ultrasound at the time of discharge from the assisted reproduction clinic, 36 and 11 of the cases were singleton and twin pregnancies, respectively. Blood samples were collected during the time span ranging from 4 to 8 weeks of gestation. The number of serial samples collected from each pregnancy ranged from 2 to 5, depending on the gestational age at which the case was discharged from the clinic. Maternal plasma ßhCG mRNA was detected in 75% (27 of 36 cases) of the singleton pregnancies and in 73% (8 of 11 cases) of the twin pregnancies. Among these cases, ßhCG mRNA was first detected in the maternal plasma collected from the singleton pregnancies at 4 weeks of gestation in 3 cases, at 5 weeks in 1 case, at 6 weeks in 19 cases, at 7 weeks in 1 case, and at 8 weeks in 3 cases. For the twin pregnancies, maternal plasma ßhCG mRNA was first detected in 6 cases at 6 weeks of gestation and in 1 case each at 7 and 8 weeks, respectively. Among all of the samples available from the singleton pregnancies during each gestational week, ßhCG mRNA was detected in 12% of samples at week 4, 33% at week 5, 63% at week 6, 60% at week 7, and 100% at week 8 (see Fig. 1A in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol52/issue2). For twin pregnancies, ßhCG mRNA was detected in none of the samples at weeks 4 and 5, in 50% of samples at week 6, in 50% at week 7, and in 100% of samples at week 8 (see Fig. 1A in the online Data Supplement). The median ßhCG mRNA concentration, when first detected, was 54 copies/mL (interquartile range, 30–230 copies/mL) and 70 (34–140) copies/mL for the singleton and twin pregnancies, respectively.
Maternal plasma hPL mRNA was detected in only 19% (7 of 36 cases) of the singleton pregnancies and in only 11% (1 of 11) of the twin pregnancies. hPL mRNA was first positively detected at 6 weeks of gestation for the twin pregnancy. For the singleton pregnancies, hPL mRNA was first detected at 4 weeks in 2 cases, at 6 and 7 weeks in 1 case each, and at 8 weeks in 3 cases. Among all of the maternal plasma samples collected from the singleton pregnancies, hPL mRNA was detected in 6%, 17%, 7%, 40%, and 33% of the samples collected at weeks 4, 5, 6, 7, and 8 of gestation, respectively (see Fig. 1B in the online Data Supplement). For the twin pregnancy samples, hPL mRNA was detected in only 1 of 12 samples collected at week 6 and in none of the other samples collected between weeks 4 and 8 of gestation (see Fig. 1B in the online Data Supplement). When first detected, the median maternal plasma hPL mRNA concentration was 41 (30–93) copies/mL in the singleton pregnancies.
To study the time profile of placental mRNA clearance from maternal plasma, we collected serial postdelivery samples from 6 pregnancies. Blood sampling for the last time point was not available for case L5. Maternal plasma concentrations of hPL mRNA after cesarean section are shown in Fig. 1
. The median hPL mRNA concentration before delivery was 9900 (3400–11 000) copies/mL. Except for case L5, the hPL mRNA concentration decreased markedly, ranging from 2.1% to 53.4% of the predelivery concentration in the first postdelivery sample, collected within 15 to 25 min of cesarean section (Fig. 1
). To estimate the apparent half-life of hPL mRNA clearance, we plotted the natural logarithms of the data from the peak hPL mRNA concentrations against time. The R2 values and slopes (–k) were calculated by linear regression using SigmaStat (Ver. 3.0; SPSS). The R2 values ranged from 0.469 to 0.922, with a median of 0.722. The apparent half-lives of hPL mRNA clearance were then calculated. The median apparent half-life was 14 min (range, 9–29 min).
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In this study, we evaluated the gestational age at which ßhCG and hPL mRNA can be detected in maternal plasma collected from early pregnancy, and we studied the kinetics of disappearance of hPL mRNA in serial postdelivery samples. In the first part of the study, women who had conceived through assisted reproductive techniques were recruited because the conception dates were accurately documented. The detection rate for ßhCG mRNA in these early-pregnancy maternal plasma samples was higher than that for hPL mRNA. This observation may be related to the concentration profiles of ßhCG and hPL in human placentas (4). We have previously shown that concentrations of both ßhCG and hPL mRNA in maternal plasma demonstrate decreasing and increasing trends, respectively, during the progression of pregnancy (2). As the maternal plasma ßhCG mRNA concentration was the highest whereas the hPL mRNA concentration was the lowest during the first trimester, it is not surprising that the detection rate of ßhCG mRNA was higher than that of hPL, up to week 8 of gestation. Furthermore, the positive detection rate of circulating ßhCG mRNA increased progressively between weeks 4 and 8 of pregnancy, but ßhCG mRNA was most reliably detected at week 8 in both singleton and twin pregnancies. On the other hand, circulating hPL mRNA was detected in only one third of the samples collected at week 8. To address the gestational age at which hPL mRNA can be reliably detected in maternal plasma, future studies would need to be extended to a later stage in pregnancy.
In the second part of the study, the clearance rate of placental mRNA in maternal plasma was evaluated. Because our previous study (2) had shown that ßhCG mRNA was detected in <10% of maternal plasma samples collected in the third trimester, we focused our current investigation on hPL mRNA. That earlier study (2) showed that hPL mRNA was no longer detectable in maternal plasma 24 h after cesarean section, and the concentrations decreased substantially within 2 h of delivery. In this investigation, we studied the apparent half-life of hPL mRNA clearance in maternal plasma in a more closely spaced group of samples. The apparent half-life was found to be in the order of minutes. It is interesting to note that the rate of hPL mRNA clearance is of the same order as that for fetal DNA clearance (7)(8). Although the mechanisms of postdelivery fetal DNA and RNA clearance from maternal plasma are unknown at present, it is interesting to speculate whether similar clearance rates between fetal DNA and hPL mRNA are the result of similar clearance mechanisms after placental separation. Future studies might address whether different placentally expressed transcripts are cleared from maternal plasma at similar time profiles.
In summary, we investigated the time profiles of the appearance of ßhCG and hPL mRNA in maternal plasma and the clearance kinetics of the latter. Unlike the study of fetal DNA, the kinetics of placental mRNA appearance and disappearance from maternal plasma may be influenced by the placental tissue concentration profile of the transcript. Thus, our study provides useful baseline data for comparison with future studies on other circulating placentally expressed transcripts in maternal plasma.
Acknowledgments
This work was supported by an Earmarked Research Grant from the Research Grants Council (CUHK 4474/03M) of the Hong Kong Special Administrative Region, and by Fondazione CARISBO Progetto Triennale-Molecular Genetics of Fetal DNA, Italy and Fondo ex-60% (to A.F.).
References
The following articles in journals at HighWire Press have cited this article:
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  C. F. Wright and H. Burton The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis Hum. Reprod. Update, January 1, 2009; 15(1): 139 - 151. [Abstract] [Full Text] [PDF]
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