SRY-specific cell free fetal DNA in maternal plasma in twin pregnancies throughout gestation

Objectives

Levels of SRY-specific cell free fetal DNA (SRY-cffDNA) in maternal plasma were investigated in twin pregnancies with two male fetuses versus one male and one female fetus and singleton male pregnancies during second and third trimester. The aim was to evaluate at which gestational age the amount of SRY-cffDNA reflects the number of fetuses and placentas respectively.

Methods

251 venous blood samples were analyzed from a total of 178 women with male or mixed-gender twin pregnancies and male singleton pregnancies in the second and the third trimester. The concentration of SRY-cffDNA was determined by quantitative real time PCR using the Y-chromosome specific SRY assay. For statistical analysis these three groups were divided into four subgroups according to their gestational age.

Results

During second trimester levels of SRY-cffDNA showed no differences between twin and singleton pregnancies. After 28 weeks SRY-cffDNA of male twin pregnancies was significantly increased compared to singleton male pregnancies and mixed-gender twin pregnancies with no differences between the latter two.

Conclusion

The level of SRY-cffDNA in maternal serum of twin pregnancies reflects the number of fetuses only during the third trimester. Hence its use as a diagnostic tool for complications related to altered SRY-cffDNA levels in twin pregnancies should be evaluated at different weeks of gestation, especially during the second trimester.     

Fluctuation of maternal and fetal free extracellular circulatory DNA in maternal plasma

OBJECTIVE: To examine whether concentrations of free extracellular fetal circulatory DNA in maternal plasma are stable or fluctuate. METHODS: Consecutive blood samples were drawn from 13 healthy nonpregnant volunteers and from 16 healthy pregnant women over 3 days. DNA was isolated from the plasma fraction and quantified by real-time polymerase chain reaction (PCR). RESULTS: In nonpregnant controls the total amount of cell free DNA fluctuated by an average of 13.5-fold. In samples obtained from pregnant women the amount of maternal cell free DNA varied by an average of 21.5-fold. Because ten of those women were pregnant with male fetuses, the concentration of free fetal DNA in these cases was determined by a real-time PCR assay for the Y chromosome. The mean variation in free fetal DNA levels in male fetuses was 2.2-fold. CONCLUSION: The degree of variation in free fetal DNA concentrations observed in this study was similar to published values, so these results imply that care should be exercised when considering quantitation of this fetal material for potential diagnostic or screening purposes.     

Prenatal diagnosis using fetal cells and free fetal DNA in maternal blood

The presence of fetal cells and free fetal DNA in maternal blood offers an exciting opportunity for the development of safe noninvasive forms of prenatal diagnosis. Research in this field has, however, also indicated that their levels in the maternal circulation are increased in certain pregnancy-related disorders, such as preeclampsia. Their closer examination may shed new light on the underlying etiology of this enigmatic disorder.     

Bovine fetal DNA in the maternal circulation: Applications and implications

Objective

The main aim of the present study was to detect bovine fetal DNA in the maternal circulation, a relatively unexplored subject in the literature.

Study design

DNA was extracted from blood of 84 primipara cows (Bos indicus) at different gestational ages (30-270 days) and from 100 adult animals (50 males and 50 non-pregnant cows). The samples were analyzed using PCR with primers for TSPY gene.

Results

Molecular results matched the fetal phenotypic gender in all 47 male and 37 female fetuses, including early pregnancy, and in control animals.

Conclusions

These results evidence a bovine transplacental fetal DNA passage.     

应用表观遗传学方法检测母体循环中游离胎儿DNA

目的:探讨表观遗传学方法检测孕妇血浆中游离胎儿DNA水平的应用价值。方法:随机选择早、中、晚期妊娠孕妇各20例,以非妊娠妇女20例为对照组。提取血浆中的总游离DNA,用甲基化特异性PCR(MSP)方法检测各组血浆标本中目的基因m-maspin(一种肿瘤抑制基因启动子的甲基化序列)、u-maspin(前者的未甲基化序列)的表达水平,再进行相对定量,对妊娠组和对照组样本均数进行统计学分析。结果:(1)m-maspin在妊娠早、中、晚期孕妇血浆中的浓度分别是非妊娠妇女血浆中浓度的1.0、1.1、1.1倍,差异无统计学意义(P>0.05);(2)u-maspin在正常非妊娠妇女血浆中未能检测到,在60例孕妇血浆中57例可检测到,它在孕妇血浆中的浓度随妊娠进展呈升高趋势,中、晚期妊娠妇女血浆中其浓度相对于早期妊娠分别为1.7倍和3.1倍,差异有统计学意义(P<0.01)。结论:m-maspin不是妊娠特异性标志物,u-maspin为妊娠特异性标志物。u-maspin基因可作为孕妇血浆中游离胎儿DNA水平变化的表观遗传学标志,相对于以胎儿性别基因作为遗传学标志的检测方法,它有助于扩大非创伤性产前诊断的临床应用范围。     

Non-invasive diagnosis of fetal sex; utilisation of free fetal DNA in maternal plasma and ultrasound

Non-invasive prenatal diagnosis is now a clinical reality, using both early ultrasound and molecular DNA methods. Technical advances in the sensitivity of the polymerase chain reaction (PCR), coupled with the finding that significant levels of fetal DNA (ffDNA) are found in maternal plasma and serum, has enabled the ready detection of paternally inherited genes or polymorphisms. Routine maternal plasma-based genotyping is now available for the determination of fetal sex and RHD blood group status (Van der Schoot et al., 2003). This review touches briefly on the ultrasound diagnoses and then focuses on the application of free ffDNA for fetal sex determination, indicating the Y-chromosome targets exploited in this strategy and the merits of their utilisation.     

Evaluation of the clinical usefulness of isolation of fetal DNA from the maternal circulation

OBJECTIVE: To assess the reliability of isolating free fetal DNA from maternal usefulness. DESIGN: Fetal DNA was isolated from plasma or serum that was either collected prospectively or from archived samples collected for the purposes of second trimester screening. METHODS: Prospective samples were collected from patients undergoing prenatal diagnostic procedures (n = 24). A second group of samples from Rhesus negative women (n = 28) were assayed in which blood had originally been collected for maternal triple serum screening. DNA was extracted from all samples and assayed for the presence of the beta-globin gene, sex-determing region Y (SRY) gene and Rh gene. All DNA sample handling and extraction was carried out by a single operator, and polymerase chain reaction (PCR) was carried out using previously published PCR primers and appropriate controls. The accuracy of results was assessed relative to the karyotype in the case of the SRY gene or cord blood phenotype in the case of the Rh gene. RESULTS: The SRY PCR results were compared to fetal cell karyotypes obtained from invasive diagnostic testing, 21 out of the 24 samples were correctly 'sexed'. The RhD PCR results were compared to fetal cord blood samples at the time of delivery, and showed both false positive and false negative results. Two RhD negative babies were genotyped as RhD positive, despite repeat analysis. CONCLUSION: It is possible to isolate fetal DNA from maternal serum. It is a potentially clinically useful technique in our laboratory and can be used to detect male fetuses, and Rh negative fetuses. To be useful in clinical practice, it is necessary to safeguard against contamination at the time of sample handling, and to use the optimal range of primers available to cover the polymorphisms present within the RhD gene. Although not robust enough yet to be used with diagnostic certainty in our hands, immense improvements in technique, probes and real-time PCR equipment make this type of diagnosis a reality in the near future.     

Effect of labor on postpartum clearance of cell-free fetal DNA from the maternal circulation

OBJECTIVE: The aim of this study was to compare the effect of mode of delivery on the postpartum clearance of cell-free fetal (cff) DNA. METHODS: Women who gave birth to a single-term male infant by any mode of delivery had blood collected on three occasions: within 3 h of birth, on day 1-2 postpartum and at 2 weeks postpartum. The SRY sequence was used as a marker of cff DNA, and was detected using conventional PCR. RESULTS: Eighty-seven women were included in the study. There were 28 women in the elective caesarean section group and 59 in the labor group. Cell-free fetal DNA was detected in 38/87 (44%) of women within 3 h of birth. There was a significant difference between the group that labored and the group that did not (54 vs 21%, p = 0.003). Twelve percent of women who labored had persistent cff DNA on day 1-2 postpartum, compared with none of the women who delivered without labor. No woman had DNA that persisted up to 2 weeks postpartum. CONCLUSION: The presence of labor increases the rate of detectable DNA in women within 3 h of birth and on day 1-2 postpartum. Postpartum clearance was completed by 2 weeks in all women tested regardless of mode of delivery.     

The best approach for early prediction of fetal gender by using free fetal DNA from maternal plasma

OBJECTIVES: Detection of free fetal DNA (ffDNA) in maternal blood during pregnancy has given rise to the possibility of developing new noninvasive approaches for early prenatal diagnosis.On a large-scale study, two protocols of real-time polymerase chain reaction (PCR) were compared in order to establish which Y-specific locus, either multicopy DYS14 or single copy SRY sequence, was the most suitable for developing a test with high diagnostic efficiency for early fetal gender assessment. The second aim was to assess whether the combination of the two detection systems could increase the performance of the prenatal test. METHODS: We analyzed 145 plasma samples from healthy pregnant women between 11 and 12 weeks of singleton gestation. For each sample, fetal gender was determined by using both protocols (DYS14 and SRY) during the same real-time PCR run. RESULTS: The data obtained by the DYS14 and SRY assays showed an efficiency in fetal gender prediction of 97.9 and 80%, respectively. It is not advisable to combine the two protocols because this association does not help in further improvements in fetal gender prediction. CONCLUSIONS: DYS14 assay is the best approach for early fetal gender assessment because it is more sensitive, accurate, and efficient than the SRY assay.     

Review: Cell-free fetal DNA in the maternal circulation as an indication of placental health and disease

In human pregnancy, the constant turnover of villous trophoblast results in extrusion of apoptotic material into the maternal circulation. This material includes cell-free (cf) DNA, which is commonly referred to as “fetal”, but is actually derived from the placenta. As the release of cf DNA is closely tied to placental morphogenesis, conditions associated with abnormal placentation, such as preeclampsia, are associated with high DNA levels in the blood of pregnant women. Over the past five years, the development and commercial availability of techniques of massively parallel DNA sequencing have facilitated noninvasive prenatal testing (NIPT) for fetal trisomies 13, 18, and 21. Clinical experience accrued over the past two years has highlighted the importance of the fetal fraction (ff) in cf DNA analysis. The ff is the amount of cell-free fetal DNA in a given sample divided by the total amount of cell-free DNA. At any gestational age, ff has a bell-shaped distribution that peaks between 10 and 20% at 10–21 weeks. ff is affected by maternal body mass index, gestational age, fetal aneuploidy, and whether the gestation is a singleton or multiple. In approximately 0.1% of clinical cases, the NIPT result and a subsequent diagnostic karyotype are discordant; confined placental mosaicism has been increasingly reported as an underlying biologic explanation. Cell-free fetal DNA is a new biomarker that can provide information about the placenta and potentially be used to predict clinical problems. Knowledge gaps still exist with regard to what affects production, metabolism, and clearance of feto-placental DNA.     

Detection of cell-free fetal DNA in maternal urine

OBJECTIVE: To evaluate the presence of cell-free fetal DNA signals in maternal urine as a potential source of material for non-invasive prenatal diagnosis. STUDY DESIGN: Patients referred to the regional fetal medicine unit who underwent prenatal diagnosis by chorionic villus sampling (CVS) were asked to give blood and urine immediately before the procedure. Maternal blood and urine were centrifuged at 10,000 g for 10 min. Plasma (1 mL) and urine (1 mL) supernatant were transferred to a clean tube and centrifuged again. The plasma (0.8 mL) and urine (0.8 mL) supernatant were removed without disturbing the cell pellet and stored at - 80 degrees C. Following DNA extraction, each sample was tested for the presence of Y chromosome associated DYS14 gene using real-time polymerase chain reaction (PCR). The total amount (maternal and fetal) of DNA in each sample was estimated using a quantitative real-time PCR assay. RESULTS: Twenty patients were enrolled in the study. CVS was performed at a median gestational age of 13 weeks (range 11 + 5 - 14 + 1). There were 12 male and 8 female fetuses, as confirmed by karyotype. Y chromosome DNA was not detected in any of the 20 samples of maternal urine, including 12 of the 20 samples in which Y chromosome DNA was detected in maternal plasma (all of whom were subsequently confirmed to be carrying a male fetus). There was considerable variation in the amount of total free DNA detected in maternal urine. CONCLUSIONS: Cell-free fetal DNA either was not present or did not amplify in maternal urine.