Tze Kin Lau

Detection and Characterization of Placental MicroRNAs in Maternal Plasma

BACKGROUND The discovery of circulating fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. MicroRNAs (miRNAs), a class of small RNAs, have been intensely investigated recently because of their important regulatory role in gene expression. Because nucleic acids of placental origin are released into maternal plasma, we hypothesized that miRNAs produced by the placenta would also be released into maternal plasma. METHODS We systematically searched for placental miRNAs in maternal plasma to identify miRNAs that were at high concentrations in placentas compared with maternal blood cells and then investigated the stability and filterability of this novel class of pregnancy-associated markers in maternal plasma. RESULTS In a panel of TaqMan MicroRNA Assays available for 157 well-established miRNAs, 17 occurred at concentrations >10-fold higher in the placentas than in maternal blood cells and were undetectable in postdelivery maternal plasma. The 4 most abundant of these placental miRNAs (miR-141, miR-149, miR-299-5p, and miR-135b) were detectable in maternal plasma during pregnancy and showed reduced detection rates in postdelivery plasma. The plasma concentration of miR-141 increased as pregnancy progressed into the third trimester. Compared with mRNA encoded by CSH1 [chorionic somatomammotropin hormone 1 (placental lactogen)], miR-141 was even more stable in maternal plasma, and its concentration did not decrease after filtration. CONCLUSION We have demonstrated the existence of placental miRNAs in maternal plasma and provide some information on their stability and physical nature. These findings open up a new class of molecular markers for pregnancy monitoring.

Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma

BACKGROUND The precise measurement of cell-free fetal DNA in maternal plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal aneuploidies and other applications. We tested the hypothesis that microfluidics digital PCR, in which individual fetal-DNA molecules are counted, could enhance the precision of measuring circulating fetal DNA. METHODS We first determined whether microfluidics digital PCR, real-time PCR, and mass spectrometry produced different estimates of male-DNA concentrations in artificial mixtures of male and female DNA. We then focused on comparing the imprecision of microfluidics digital PCR with that of a well-established nondigital PCR assay for measuring male fetal DNA in maternal plasma. RESULTS Of the tested platforms, microfluidics digital PCR demonstrated the least quantitative bias for measuring the fractional concentration of male DNA. This assay had a lower imprecision and higher clinical sensitivity compared with nondigital real-time PCR. With the ZFY/ZFX assay on the microfluidics digital PCR platform, the median fractional concentration of fetal DNA in maternal plasma was > or =2 times higher for all 3 trimesters of pregnancy than previously reported. CONCLUSIONS Microfluidics digital PCR represents an improvement over previous methods for quantifying fetal DNA in maternal plasma, enabling diagnostic and research applications requiring precise quantification. This approach may also impact other diagnostic applications of plasma nucleic acids, e.g., in oncology and transplantation.

Clinicalapplicationofmassivelyparallelsequencing-basedprenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11105 pregnancies with mixed risk factors

To report the performance of massively parallel sequencing (MPS) based prenatal noninvasive fetal trisomy test based on cell‐free DNA sequencing from maternal plasma in a routine clinical setting in China.

Non‐invasive prenatal testing for trisomies 21, 18 and 13: clinical experience from 146 958 pregnancies

To report the clinical performance of massively parallel sequencing‐based non‐invasive prenatal testing (NIPT) in detecting trisomies 21, 18 and 13 in over 140 000 clinical samples and to compare its performance in low‐risk and high‐risk pregnancies.

Noninvasive prenatal diagnosis of common fetal chromosomal aneuploidies by maternal plasma DNA sequencing

Objective: To develop a new bioinformatic method in the noninvasive prenatal identification of common fetal aneuploidies using massively parallel sequencing on maternal plasma. Methods: Massively parallel sequencing was performed on plasma DNA samples from 108 pregnant women (median gestation: 12+5 week) immediately before chorionic villus sampling (CVS) or amniocentesis. Data were analysed using a novel z-score method with internal reference chromosome. The diagnostic accuracies of the fetal karyotyping status were compared against two previously reported z-score methods – one without adjustment and the other with GC correction. Results: A total of 32 cases with fetal aneuploidy were confirmed by conventional karyotyping, including 11 cases of Trisomy 21, 10 cases of Trisomy 18, 2 cases of Trisomy 13, 8 cases of Turner syndrome (45, XO) and one case of Klinefelter syndrome (47, XXY). Using the z-score method without reference adjustment, the detection rate for Trisomy 21, Trisomy 18, Trisomy 13, Turner syndrome, and Klinefelter’s syndrome is 100%, 40%, 0%, 88% and 0% respectively. Using the z-score method with GC correction, the detection rate increased to 100% for Trisomy 21, 90% for Trisomy 18, 100% for Trisomy 13. By using the z-score method with internal reference, the detection rate increased to 100% for all aneuploidies. The false positive rate was 0% for all three methods. Conclusion: This massively parallel sequencing-based approach, combined with the improved z-score test methodology, enables the prenatal diagnosis of most common aneuploidies with a high degree of accuracy, even in the first trimester of pregnancy.

Secondary findings from non‐invasive prenatal testing for common fetal aneuploidies by whole genome sequencing as a clinical service

To report secondary or additional findings arising from introduction of non‐invasive prenatal testing (NIPT) for aneuploidy by whole genome sequencing as a clinical service.

Impact of First Childbirth on Changes in Women’s Preference for Mode of Delivery: Follow-up of a Longitudinal Observational Study

BACKGROUND A womans childbirth experience has an influence on her future preferred mode of delivery. This study aimed to identify determinants for women who changed from preferring a planned vaginal birth to an elective cesarean section after their first childbirth. METHODS This prospective longitudinal observational study involved two units that provide obstetric care in Hong Kong. A mail survey was sent to 259 women 6 months after their first childbirth. These women had participated in a longitudinal cohort study that examined their preference for elective cesarean section in the antenatal period of their first pregnancies. Univariate and multivariate analyses were performed to identify determinants for women who changed from preferring vaginal birth to elective cesarean section. RESULTS Twenty-four percent (23.8%, 95% CI 18.4-29.3) of women changed from preferring vaginal birth to elective cesarean section after their first childbirth. Determinants found to be positively associated with this change included actual delivery by elective cesarean section (OR 106.3, 95% CI 14.7-767.4) intrauterine growth restriction (OR 19.5, 95% CI 1.1-353.6), actual delivery by emergency cesarean section (OR 8.4, 95% CI 3.4-20.6), higher family income (OR 3.2, 95% CI 1.1-8.8), use of epidural analgesia (OR 2.6, 95% CI 1.0-6.8), and higher trait anxiety score (OR 1.1, 95% CI 1.0-1.3). The most important reason for women who changed from preferring vaginal birth to elective cesarean section was fear of vaginal birth (24.4%). CONCLUSIONS A significant proportion of women changed their preferred mode of delivery after their first childbirth. Apart from reducing the number of cesarean sections in nulliparous women, prompt provision of education to women who had complications and investigations into fear factors during vaginal birth might help in reducing womens wish to change to elective cesarean section.

Pregnancy outcome after successful external cephalic version for breech presentation at term.

OBJECTIVE Our purpose was to review the outcome of pregnancies after external cephalic version at term, in particular the incidence and indications of intrapartum cesarean section after successful external cephalic version. STUDY DESIGN A prospective study was performed of 241 term pregnancies that had a total of 243 external cephalic versions. Each case with successful external cephalic version was matched to two control cases with cephalic presentation to compare pregnancy outcome. RESULTS External cephalic version was successful in 169 attempts (69.5%), of which 7 (4.1%) reverted to breech presentation. There was one case of abruptio placentae and eight cases (3.3%) of transient fetal bradycardia after the procedure. Among those who had a successful external cephalic version, the incidence of intrapartum cesarean section was 16.9%, which was 2.25 times higher than that of the control group (p < 0.005). This large number of abdominal deliveries was due to a significantly higher incidence of fetal distress and dystocic labor. The incidence of augmentation of labor was also significantly higher in the study group (37.7% vs 27.6%, p < 0.05). CONCLUSION Pregnancies after a successful external cephalic version at term are not the same as those with cephalic presentation. They are at higher risk of both dystocic labor and fetal distress and therefore require close intrapartum monitoring.

A method for noninvasive detection of fetal large deletions/ duplications by low coverage massively parallel sequencing

To report the feasibility of fetal chromosomal deletion/duplication detection using a novel bioinformatic method of low coverage whole genome sequencing of maternal plasma.

Discordant results between fetal karyotyping and non-invasive prenatal testing by maternal plasma sequencing in a case of uniparental disomy 21 due to trisomic rescue

Uniparental disomy (UPD) is an uncommon chromosome condition, but UPD involving chromosome 21 is rarely reported. We reported here a case who had first trimester screening test for Down syndrome, chorionic villus sampling for fetal karyotyping, quantitative fluorescence polymerase chain reaction (QF‐PCR), as well as non‐invasive prenatal testing (NIPT) by maternal plasma sequencing. There were discordant results between fetal karyotyping and NIPT due to UPD 21combined with confined placental mosaicism of trisomy 21. This demonstrated that it is possible to detect placental mosaicism by NIPT, but further studies are required to confirm its sensitivity. Therefore, all positive NIPT results must be confirmed by conventional invasive test and karyotyping. QF‐PCR has the additional benefit in diagnosing UPD.