Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples.

BACKGROUND: DNA methylation plays crucial roles in epigenetic gene regulation in normal development and disease pathogenesis. Efficient and accurate quantification of DNA methylation at single base resolution can greatly advance the knowledge of disease mechanisms and be used to identify potential biomarkers. We developed an improved pipeline based on reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide quantification of DNA methylation at single base resolution. A selection of two restriction enzymes (TaqαI and MspI) enables a more unbiased coverage of genomic regions of different CpG densities. We further developed a highly automated software package to analyze bisulfite sequencing results from the Solexa GAIIx system. RESULTS: With two sequencing lanes, we were able to quantify ~1.8 million individual CpG sites at a minimum sequencing depth of 10. Overall, about 76.7% of CpG islands, 54.9% of CpG island shores and 52.2% of core promoters in the human genome were covered with at least 3 CpG sites per region. CONCLUSIONS: With this new pipeline, it is now possible to perform whole-genome DNA methylation analysis at single base resolution for a large number of samples for understanding how DNA methylation and its changes are involved in development, differentiation, and disease pathogenesis.

DNMT1 and AIM1 Imprinting in human placenta revealed through a genome-wide screen for allele-specific DNA methylation.

BACKGROUND: Genomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. Many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. Such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. This is particularly relevant for the placenta. Study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. We describe here a new strategy to study allele-specific DNA methylation in the human placenta for the discovery of novel imprinted genes. RESULTS: Using this methodology, we confirmed 16 differentially methylated regions (DMRs) associated with known imprinted genes. We chose 28 genomic regions for further testing and identified two imprinted genes (DNMT1 and AIM1). Both genes showed maternal allele-specific methylation and paternal allele-specific transcription. Imprinted expression for AIM1 was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse. CONCLUSIONS: Our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. Nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. Their identification may help us better understand embryonic and fetal development.

Risk factors and outcomes of uterine rupture in Singapore: Emerging trends.

INTRODUCTION: Uterine rupture is uncommon but has catastrophic implications on the pregnancy. A scarred uterus and abnormal placentation are known contributory factors. The aim of our study was to review the contributing factors, clinical presentation, complications and management of uterine rupture in our population in light of the changing nature of modern obstetric practices. METHODS: A retrospective observational study was conducted at KK Women's and Children's Hospital by studying proven cases of uterine rupture in the period between January 2003 and December 2014. These cases were analysed according to their past history, clinical presentation, complications, management and outcome. RESULTS: A total of 48 cases of proven uterine rupture were identified. The incidence of uterine rupture was 1 in 3,062 deliveries. The ratio of scarred uterus rupture to unscarred uterus rupture was approximately 3:1. The most common factor was previous lower segment caesarean section for the scarred group, followed by a history of laparoscopic myomectomy. Abdominal pain was the common clinical presentation in the antenatal period, while abnormal cardiotocography findings were the most common presentation in intrapartum rupture. CONCLUSION: There is a notable shift in the trend of uterine rupture cases given the increasing use of laparoscopic myomectomy and elective caesarean sections. While ruptures from these cases were few, their presentation in the antenatal period calls for diligent monitoring with informed patient involvement in their pregnancy care.

DORSCON Orange: An Approach to Challenges in a Busy Antenatal Diagnostic Centre in the Midst of a Global Pandemic.

The WHO declared the coronavirus disease 2019 (COVID-19) a global pandemic on 11 March 2020. Lessons from SARS epidemic led Singapore to develop stringent infection control protocols in preparation for future pandemics. However, unlike SARS, COVID-19 appears to be more transmissible and is predicted to continue for longer. As of 14 June 2020, there have been 40,197 positive cases with 26 deaths in Singapore, and KK Women's and Children's Hospital (KKH) has managed a total of 73 cases. Obstetrics ultrasound is an indispensable medical service and must continue to operate during a pandemic. A key balance must be struck between keeping patients and healthcare workers safe while being able to provide quality and prompt obstetric care. Our Antenatal Diagnostic Centre (ADC) in KKH developed new strategies to adapt to the pandemic when the national Disease Outbreak Response System Condition (DORSCON) was raised from yellow to orange on 7 February 2020. In this paper, we discuss our clinical workflow to reduce the risk of transmission amongst patients and staff while minimising disruption to our services.

Angiogenic factors during pregnancy in Asian women with elevated blood pressure in early pregnancy and the risk of preeclampsia: a longitudinal cohort study.

OBJECTIVE: It remains unclear what roles placenta-originated angiogenic factors play in the pathogenesis of preeclampsia among hypertensive women. We compared maternal soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) levels throughout pregnancy in women with normal blood pressure (BP), elevated BP and hypertension in early pregnancy and their risks of developing preeclampsia. DESIGN: A prospective cohort study. SETTING: KK Women's and Children's Hospital, Singapore. PARTICIPANTS: 923 women with singleton pregnancy <14 weeks of gestation were included in the prospective Neonatal and Obstetrics Risks Assessment cohort between September 2010 and October 2014. Systolic, diastolic, mean arterial blood pressure (MAP) were measured at 11-14 weeks. PRIMARY AND SECONDARY OUTCOMES: Maternal serum sFlt-1, PlGF and sFlt-1/PlGF ratio were tested at 11-14, 18-22, 28-32 and 34 weeks onwards of gestation. Preeclampsia was main pregnancy outcome. RESULTS: Women were divided based on their BP in early pregnancy: normal (n=750), elevated BP (n=98) and hypertension (n=75). Maternal sFlt-1 levels and sFlt-1/PlGF ratios were higher in hypertensive women throughout pregnancy, but maternal PlGF levels were not significantly lower. Rise in maternal systolic, diastolic BP and MAP at 11-14 weeks were significantly associated with higher sFlt-1/PlGF ratios during pregnancy. A 10 mm Hg increase in MAP was associated with a 5.6-fold increase in risk of preterm preeclampsia and a 3.3-fold increase in risk of term preeclampsia, respectively. CONCLUSION: Women with elevated BP in early pregnancy already had a higher sFlt-1/PlGF ratio in early gestation and throughout pregnancy, and an increased risk of preeclampsia. In contrast, PlGF levels in these women remained normal.

Does Low Birth Weight Vary Geospatially in Singapore?

INTRODUCTION: Low birth weight (LBW, <2500 g) is an important risk factor for perinatal mortality and morbidity. We performed the first geospatial study of LBW in Singapore, with focus on the public sector and analysis of the national planning areas. MATERIALS AND METHODS: A dataset of 24,615 singleton deliveries from 2012 to 2014 was obtained from the largest maternity hospital in Singapore. Maternal residences were identified with 28 planning areas according to postal code. Multiple logistic regression was used to examine associations between LBW rates and planning areas. Moran's I statistic was used to test for geospatial clustering of LBW rates among planning areas. RESULTS: The LBW rate across planning areas ranged from 5.3 to 11.5 per 100 live births (median, 8.4). High LBW rates were associated with: 1) a lower individual socioeconomic status, 2) non-compliance to antenatal visits, and 3) biological factors such as maternal hypertension, low body mass index and Indian race. Moran's statistic indicated no geospatial clustering of LBW rates among the 28 planning areas (P = 0.12). LBW rates were moderately correlated with the Socioeconomic Disadvantage Index (r = 0.58) but uncorrelated with distance travelled to hospital (r = -0.08). CONCLUSION: There was no evidence of clustering of LBW rates among planning areas in Singapore that would indicate inequitable distribution of health resources among planning areas. The 2 areas showing the highest rates of LBW infants were Outram and Bukit Merah. We recommend targeted health interventions and outreach programmes to encourage antenatal visits in these areas.

Fetoscopic laser photocoagulation in twin-to-twin transfusion syndrome: experience from a single institution.

INTRODUCTION: Twin-to-twin transfusion syndrome (TTTS) is the most common serious complication of monochorionic (MC) twin pregnancies, with perinatal mortality rates of up to 90% if untreated. This study aimed to review the perinatal and perioperative outcomes of MC twin pregnancies treated for TTTS by fetoscopic laser photocoagulation (FLP) since its introduction at KK Women's and Children's Hospital (KKH), Singapore, in 2011. METHODS: This was a retrospective review of five consecutive patients who underwent FLP of placental anastomoses for TTTS at KKH from June 2011 to March 2014. FLP was offered to patients who were diagnosed with TTTS of at least Quintero Stage II before 26 weeks of gestation. The main outcome measures were perioperative complications and perinatal survival rates. RESULTS: Five sets of MC twin pregnancies underwent FLP during the study period - three pregnancies were diagnosed with Stage III TTTS and two pregnancies with Stage II TTTS. Median gestational ages at initial presentation, laser photocoagulation and delivery were 19 (range 17-20) weeks, 20 (range 19-23) weeks and 29 (range 28-34) weeks, respectively. One patient had bleeding into the amniotic cavity intraprocedurally. Overall, the perinatal survival rate, double-infant survival rate and survival rate for at least one twin were 60% (6/10 fetuses), 40% (2/5 twins) and 80% (4/5 twins), respectively. CONCLUSION: FLP is a feasible treatment for TTTS, with minimal maternal complications. Perinatal survival rates of this patient group that was managed at our centre were comparable to those of international centres.

Correlation of cord blood telomere length with birth weight.

BACKGROUND: Intrauterine growth restriction affects 3% of newborns; and the lightest 10% of whom are classified as small for gestational age (SGA). These low-birth weight newborns are at increased risk of neonatal morbidity such as hypoxia and hypoglycaemia. In later life, they are at higher risk of several age-related diseases such as cardiovascular and metabolic disorders and dementia. As having short telomeres is also associated with these diseases, we tested if these newborns might already start with shorter telomeres at birth. FINDINGS: Relative telomere lengths were determined using quantitative real-time PCR in cord blood samples from 195 newborns of Chinese ancestry. Based on the telomere length normalised to a single copy gene and a reference DNA sample as internal control, we found statistically significant correlations between relative telomere length and both unadjusted and gestational age-adjusted birth weight, with the lighter newborns having shorter telomeres. The SGA birth weight group comprising the bottom 10% of the samples also had the shortest telomeres compared to the medium and heaviest birth weight groups. CONCLUSIONS: Our results indicate that there is reduction of cord blood telomere length for newborns with lower birth weight.

Gender-Specific Reference Charts of Fetal Head Circumference in a Singaporean Population.

INTRODUCTION: With the global outbreak of Zika virus and its association with microcephaly, an up-to-date fetal head circumference (HC) nomogram is crucial to offer a reference standard in order to make an accurate diagnosis. This study was conducted to revise the local fetal HC nomogram. MATERIALS AND METHODS: In this retrospective study, ultrasound data was used for construction of the fetal HC nomogram from a total of 6155 pregnancies in the ethnic Chinese population with low risk profile at KK Women's and Children's Hospital over a 10-year period. Regression model was fitted to calculate the mean and standard deviation of HC at each gestational age (GA). Comparison of HC between ethnic groups (no significant differences) and genders were made. The revised chart was compared with another commonly used reference chart (Hadlock). In an independent test population, different reference charts were used to estimate number of cases with microcephaly. RESULTS: A statistically significant difference of HC between the genders was observed across all gestational ages. Gender-specific reference charts and equation were computed. Our revised fetal HC chart showed a different distribution from the Hadlock chart. Compared with the gender-specific charts, the Hadlock HC chart would significantly under-report microcephaly cases in male fetuses, and tend to over-report in female fetuses. CONCLUSION: This study provides a new set of gender-specific fetal HC charts in the Singaporean population for antenatal ultrasound surveillance of microcephaly.