Genome-wide detection of cytosine methylation by single molecule real-time sequencing.

5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human-mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.

Detection and characterization of jagged ends of double-stranded DNA in plasma.

Cell-free DNA in plasma has been used for noninvasive prenatal testing and cancer liquid biopsy. The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have attracted much recent interest, leading to the emerging field of cell-free DNA fragmentomics. However, one aspect of plasma DNA fragmentomics as to whether double-stranded plasma molecules might carry single-stranded ends, termed a jagged end in this study, remains underexplored. We have developed two approaches for investigating the presence of jagged ends in a plasma DNA pool. These approaches utilized DNA end repair to introduce differential methylation signals between the original sequence and the jagged ends, depending on whether unmethylated or methylated cytosines were used in the DNA end-repair procedure. The majority of plasma DNA molecules (87.8%) were found to bear jagged ends. The jaggedness varied according to plasma DNA fragment sizes and appeared to be in association with nucleosomal patterns. In the plasma of pregnant women, the jaggedness of fetal DNA molecules was higher than that of the maternal counterparts. The jaggedness of plasma DNA correlated with the fetal DNA fraction. Similarly, in the plasma of cancer patients, tumor-derived DNA molecules in patients with hepatocellular carcinoma showed an elevated jaggedness compared with nontumoral DNA. In mouse models, knocking out of the Dnase1 gene reduced jaggedness, whereas knocking out of the Dnase1l3 gene enhanced jaggedness. Hence, plasma DNA jagged ends represent an intrinsic property of plasma DNA and provide a link between nuclease activities and the fragmentation of plasma DNA.

Integrative single-cell and cell-free plasma RNA transcriptomics elucidates placental cellular dynamics.

The human placenta is a dynamic and heterogeneous organ critical in the establishment of the fetomaternal interface and the maintenance of gestational well-being. It is also the major source of cell-free fetal nucleic acids in the maternal circulation. Placental dysfunction contributes to significant complications, such as preeclampsia, a potentially lethal hypertensive disorder during pregnancy. Previous studies have identified significant changes in the expression profiles of preeclamptic placentas using whole-tissue analysis. Moreover, studies have shown increased levels of targeted RNA transcripts, overall and placental contributions in maternal cell-free nucleic acids during pregnancy progression and gestational complications, but it remains infeasible to noninvasively delineate placental cellular dynamics and dysfunction at the cellular level using maternal cell-free nucleic acid analysis. In this study, we addressed this issue by first dissecting the cellular heterogeneity of the human placenta and defined individual cell-type-specific gene signatures by analyzing more than 24,000 nonmarker selected cells from full-term and early preeclamptic placentas using large-scale microfluidic single-cell transcriptomic technology. Our dataset identified diverse cellular subtypes in the human placenta and enabled reconstruction of the trophoblast differentiation trajectory. Through integrative analysis with maternal plasma cell-free RNA, we resolved the longitudinal cellular dynamics of hematopoietic and placental cells in pregnancy progression. Furthermore, we were able to noninvasively uncover the cellular dysfunction of extravillous trophoblasts in early preeclamptic placentas. Our work showed the potential of integrating transcriptomic information derived from single cells into the interpretation of cell-free plasma RNA, enabling the noninvasive elucidation of cellular dynamics in complex pathological conditions.

Impact of preimplantation genetic testing for aneuploidy on obstetrical practice.

PURPOSE OF REVIEW: To provide updated information on preimplantation genetic testing for aneuploidy (PGT-A), focusing on its implications on prenatal diagnostic approaches after transferal of mosaic aneuploid embryos. RECENT FINDINGS: PGT-A is a technology to screen for chromosome aneuploidy or major chromosome structural rearrangement in embryos before implantation using different cytogenetic analyses. PGT-A has been shown to reduce the negative effect of increasing maternal age on in-vitro fertilization (IVF) outcomes. PGT-A also encourages clinicians and patients to accept single embryo transfer after IVF-PGT-A to reduce the chance of multiple pregnancies. However, mosaic aneuploid embryo may be encountered following PGT-A. Recent discussions have focused on the consideration of transferal of mosaic aneuploid embryos in couples with no euploid embryo following multiple trials of IVF-PGT-A. A risk score for each chromosome has been developed to prioritize which mosaic embryos should be considered for transfer. However, there is a lack of long-term outcome data following mosaic aneuploid embryo transfer. SUMMARY: Recent advances in PGT-A increase the detection of embryos with mosaicism, which is associated with an increased risk of miscarriage, fetal mosaic aneuploidy, and uniparental disomy. Strategy in prenatal diagnostic testing after mosaic aneuploid embryo transfer is discussed in this review.

Contingent Screening for Small by Weight for Gestational Age Neonates.

Effective screening for small for gestational age neonates (SGA), in the absence of preeclampsia, can be accomplished using a contingent screening method. The basis for the contingent model is a combined assessment at 19-24 weeks gestation to stratify patients according to their risk. We can then identify prenatally over 90% of SGA neonates for a false positive rate of 10%.

Comparison of uterine artery Doppler measurements at 6 weeks of pregnancy after IVF between pregnancies that resulted in miscarriage and ongoing pregnancies.

OBJECTIVE: To compare uterine artery pulsatility index (UTPI) at 6 weeks of pregnancy following in vitro fertilization (IVF) and embryo transfer (ET) between clinical pregnancies that resulted in a miscarriage and those that were ongoing beyond 12 weeks. METHODS: A prospective observational study was conducted in an IVF unit at Prince of Wales Hospital, Hong Kong, between December 1, 2017 and December 31, 2019. UTPI was measured at 6 weeks of pregnancy among women who conceived following IVF/ET. RESULTS: Among 153 participants, 22 (14.4%) had a miscarriage whereas 131 (85.6%) had an ongoing pregnancy beyond 12 weeks. Median UTPI in pregnancies that ended in a miscarriage was significantly lower than those that progressed beyond 12 weeks (2.1, IQR 1.9-2.4 vs 2.50, IQR 2.2-2.9, respectively; P<0.001). The likelihood of the pregnancy ending in a miscarriage when the UTPI was above the 75th percentile (>2.9), between the 25th-75th percentiles (2.2-2.9), and below the 25th percentile (<2.2) was 0%, 13.2%, and 27.7%, respectively (P=0.001). CONCLUSIONS: IVF pregnancies that resulted in a miscarriage were associated with reduced resistance to uterine artery blood flow at 6 weeks of pregnancy.

Protocol for measurement of mean arterial pressure at 10-40weeks' gestation.

The study aimed to identify the simplest protocol for the measurement of mean arterial pressure (MAP) across 10-40weeks' gestation. 2726 women with uncomplicated singleton pregnancy attending for their routine hospital visit between 10 and 40weeks' gestation were recruited prospectively. The blood pressure (BP) was measured according to the National Heart Foundation of Australia (NHFA) protocol using automated devices. Linearizing regression models were determined for MAP derived from single, repeat and average measurements taken in the left and right arms using the same polynomial power of the best fit model determined using the NHFA protocol. Z-scores were used to compare the differences between the smoothed 50th percentiles. The first measurements taken in the left and right arms were on average 0.15SD and 0.12SD, respectively, higher than those obtained from the NHFA protocol. The second measurements taken in the left and right arms were both 0.26SD lower than the first measurement taken in the same arm and these values were lower than those from the NHFA protocol. The median MAP determined by the protocol of the average of two measurements taken in both arms was similar to the median MAP determined using the NHFA protocol (Z-score 0.0194SD). MAP derived by the average of two measurements in both arms had a quadratic relationship with gestation, with the measurement being the lowest in the mid-trimester. In conclusion, our study has demonstrated that at 10-40weeks' gestation, BP recordings can be obtained by a simpler protocol using the average of two measurements in both arms.

The use of ultrasound and other markers for early detection of preeclampsia.

Preeclampsia (PE) is a multisystem disorder of pregnancy classically characterized with the onset of hypertension after 20 weeks gestation in the presence of proteinuria. PE typically affects 2-8% of pregnancies and is a leading cause of maternal and perinatal morbidity and mortality. This article reviews the most effective biomarkers used in first trimester screening for PE. It explores their use both in isolation and as part of an algorithm to yield the best detection rates. Screening by a combination of maternal risk factors, uterine artery Doppler, mean arterial pressure, maternal serum PAPP-A and PlGF can identify about 75% of cases of preterm PE for a false-positive rate of 10%. By identifying these patients at high risk for PE, appropriately tailored antenatal surveillance can be instigated and prophylactic pharmacological interventions can be prescribed to improve placentation and ultimately, the outcome for both the mother and fetus.