Fiona L. Mackie

Exome Sequencing in Fetuses with Structural Malformations

Prenatal diagnostic testing is a rapidly advancing field. An accurate diagnosis of structural anomalies and additional abnormalities in fetuses with structural anomalies is important to allow “triage” and designation of prognosis. This will allow parents to make an informed decision relating to the pregnancy. This review outlines the current tests used in prenatal diagnosis, focusing particularly on “new technologies” such as exome sequencing. We demonstrate the utility of exome sequencing above that of conventional karyotyping and Chromosomal Microarray (CMA) alone by outlining a recent proof of concept study investigating 30 parent-fetus trios where the fetus is known to have a structural anomaly. This may allow the identification of pathological gene anomalies and consequently improved prognostic profiling, as well as excluding anomalies and distinguishing between de novo and inherited mutations, in order to estimate the recurrence risk in future pregnancies. The potential ethical dilemmas surrounding exome sequencing are also considered, and the future of prenatal genetic diagnosis is discussed.

Placental dysfunction is associated with altered microRNA expression in pregnant women with low folate status

Scope Low maternal folate status during pregnancy increases the risk of delivering small for gestational age (SGA) infants, but the mechanistic link between maternal folate status, SGA, and placental dysfunction is unknown. microRNAs (miRNAs) are altered in pregnancy pathologies and by folate in other systems. We hypothesized that low maternal folate status causes placental dysfunction, mediated by altered miRNA expression. Methods and results A prospective observational study recruited pregnant adolescents and assessed third trimester folate status and placental function. miRNA array, QPCR, and bioinformatics identified placental miRNAs and target genes. Low maternal folate status is associated with higher incidence of SGA infants (28% versus 13%, p < 0.05) and placental dysfunction, including elevated trophoblast proliferation and apoptosis (p < 0.001), reduced amino acid transport (p < 0.01), and altered placental hormones (pregnancy‐associated plasma protein A, progesterone, and human placental lactogen). miR‐222‐3p, miR‐141‐3p, and miR‐34b‐5p were upregulated by low folate status (p < 0.05). Bioinformatics predicted a gene network regulating cell turnover. Quantitative PCR demonstrated that key genes in this network (zinc finger E‐box binding homeobox 2, v‐myc myelocytomatosis viral oncogene homolog (avian), and cyclin‐dependent kinase 6) were reduced (p < 0.05) in placentas with low maternal folate status. Conclusion This study supports that placental dysfunction contributes to impaired fetal growth in women with low folate status and suggests altered placental expression of folate‐sensitive miRNAs and target genes as a mechanistic link.

Chromosomal microarray analysis allows prenatal detection of low level mosaic autosomal aneuploidy

Chromosomal microarray analysis (CMA) has demonstrated great clinical utility in diagnosing pathogenic copy number variants (CNVs) associated with paediatric developmental disorders and thus is replacing conventional karyotype analysis in this subset of patients. Although its application in prenatal diagnosis is currently less advanced, it is being evaluated by international multicentre studies, which are continuing to illustrate its capability at detecting both microscopic and submicroscopic CNVs. The routine use of CMA in prenatal diagnosis may be dependent upon international or national consensuses relating to the choice of platform so as to maximise the detection of pathogenic CNVs, whilst minimising the detection of variants of unknown significance (VOUS). In addition, robust health economic analysis will also be required. We recently published our prospective cohort study using a focussed bacterial artificial chromosome microarray, which examined pregnancies with abnormal ultrasound scan findings. This noted an excess detection rate of CNVs of 4.1% over conventional karyotype analysis. When examined in the context of a systematic review of the literature (using a variety of microarray platforms), the overall detection rate of CNVs (both pathogenic and VOUS) in pregnancies with ultrasound anomalies was 10%. Further studies such as this, along with the attitudes of patients and health professionals, will help ascertain the feasibility and desirability of replacing conventional karyotype analysis with CMA in prenatal diagnosis. One limitation of CMA is its inability to detect some instances of mosaicism. The level at which mosaicism may be detected by CMA will be dependent on the size of the mosaic imbalance, whether the imbalance is a gain or loss of genomic material, the design of the microarray platform and whether DNA has been extracted from cultured or uncultured cells. Current literature states that CMA can detect constitutional postnatal mosaicism on an oligonucleotide platform at a level of between 8% and 10%. The sensitivity of CMA for the detection of prenatal mosaicism has been less extensively investigated. Scott S et al. used oligonucleotide CMA and artificially derived whole chromosome and segmental aneuploidies using DNA from prenatal specimens to detect mosaicism as low as 10% and 20% to 30%, respectively. We report low level mosaic autosomal aneuploidy detected in two prenatal cases using oligonucleotide CMA to investigate congenital anomalies, where fetal DNA had been extracted from uncultured amniocytes. Patient and reference DNAs were differentially fluorescently labelled and competitively hybridised to International Standards for Cytogenomic Arrays oligonucleotide microarrays (CytoChip International Standards for Cytogenomic Arrays 8 × 60K v2.0, BlueGnome Ltd, Cambridge, UK).

Cell‐free fetal DNA‐based noninvasive prenatal testing of aneuploidy

Noninvasive prenatal testing (NIPT) uses cell‐free fetal DNA (cffDNA) to test for aneuploidy, as opposed to noninvasive prenatal diagnosis (NIPD), which uses cffDNA to diagnose fetal sex, Rhesus D status and monogenic disorders. This classic review focuses on screening for aneuploidy. NIPT is a screening test and needs confirmatory invasive testing in cases of a high‐risk (positive) result. NIPT demonstrates high sensitivities and specificities according to our recent meta‐analysis, although it is less accurate for Trisomy 18, Trisomy 13, Monosomy X and sex chromosomal aneuploidies than for Trisomy 21. It is imperative that the implications of false positive and false negative results are investigated and considered in a clinical context.

Brain-injured Survivors of Monochorionic Twin Pregnancies Complicated by Single Intrauterine Death: MR Findings in a Multicenter Study

Purpose To describe and classify the range of brain injuries present at prenatal, in-utero magnetic resonance (MR) imaging in co-twin survivors of monochorionic (MC) twin pregnancies complicated by single intrauterine death (SIUD). Materials and Methods This retrospective, observational study from six tertiary fetal medicine centers that perform tertiary-level prenatal in-utero MR studies reviewed cases in which prenatal in-utero MR imaging had shown a brain injury in a surviving co-twin of a twin pregnancy with a MC component complicated by SIUD. Results Forty-two surviving MC twins were described. The primary distinction of brain abnormalities was into nonfocal and focal lesions. The nonfocal lesions included periventricular leukomalacia (group 1; two fetuses), generalized encephalomalacia (group 2; nine fetuses), posterior encephalomalacia (group 3; seven fetuses), and bilateral parasagittal and perisylvian injury (group 4; three fetuses). The focal lesions included nonhemorrhagic lesions (group 5; 14 fetuses) and hemorrhagic lesions (group 6; seven fetuses). Focal brain lesions were more likely to be found in the surviving MC pregnancies complicated by twin-twin transfusion syndrome (TTTS) (odds ratio, 2.4; 95% confidence interval: 1.3, 18.5; P = .01) and in fetuses that underwent an obstetric intervention (odds ratio, 2.8; 95% confidence interval: 1.8, 23.6; P = .006). Conclusion Brain injury of the surviving co-twin after SIUD in MC pregnancies is usually of ischemic origin and spares the brainstem and cerebellum. Focal brain lesions are more frequent in pregnancies complicated by TTTS or in those where an intervention has been performed.

Early prognostic factors of outcomes in monochorionic twin pregnancy: systematic review and meta-analysis

BACKGROUND: Monochorionic twin pregnancies are high‐risk, however at present, no screening test is available to predict which monochorionic twin pregnancy will develop complications. OBJECTIVE: We sought to assess ability of first‐trimester pregnancy–related factors (ultrasound measurements, maternal characteristics, biomarkers) to predict complications in monochorionic twin pregnancies. DATA SOURCES: Data sources were MEDLINE, Embase, ISI Web of Science, CINAHL, the Cochrane Central Registration of Controlled Trials and Research Registers, and Google Scholar, from inception to May 12, 2017. Gray literature and bibliographies of articles were checked. STUDY ELIGIBILITY CRITERIA: Studies that reported ultrasound measurements, maternal characteristics, or potential biomarkers, measured in the first trimester in monochorionic‐diamniotic twin pregnancies, where the potential prognostic ability between the variable and twin‐twin transfusion syndrome, growth restriction, or intrauterine fetal death could be assessed, were included. STUDY APPRAISAL AND SYNTHESIS METHODS: Quality assessment was evaluated using the Strengthening the Reporting of Observational Studies in Epidemiology checklist by 2 reviewers independently. For meta‐analysis, odds ratios using a random effects model, or standardized mean difference were calculated. If a moderate association was found, the prognostic ability was evaluated by calculating the sensitivity and specificity. Risk of heterogeneity was reported as I2 and publication bias was visually assessed by funnel plots and quantitatively by Egger test. RESULTS: In all, 48 studies were eligible for inclusion. Twenty meta‐analyses could be performed. A moderate association was demonstrated in 3 meta‐analyses, between: nuchal translucency >95th centile in one/both fetuses and twin‐twin transfusion syndrome (odds ratio, 2.29 [95% confidence interval, 1.05–4.96], I2 = 6.6%, 4 studies, 615 pregnancies); crown‐rump length discordance ≥10% and twin‐twin transfusion syndrome (odds ratio, 2.43 [95% confidence interval, 1.13–5.21], I2 = 14.1%, 3 studies, 708 pregnancies); and maternal ethnicity and twin‐twin transfusion syndrome (odds ratio, 2.12 [95% confidence interval, 1.17–3.83], I2 = 0.0%, 5 studies, 467 pregnancies), but none demonstrated a prognostic ability for any outcome under investigation. CONCLUSION: It is not currently possible to predict adverse outcomes in monochorionic twin pregnancies. We have revealed a lack of research investigating first‐trimester biomarkers in monochorionic twin pregnancies. Different assessment methods and definitions of each variable and outcome were an issue and this highlights the need for a large cohort study to evaluate these factors.

The prediction, diagnosis and management of complications in monochorionic twin pregnancies: the OMMIT (Optimal Management of Monochorionic Twins) study

BackgroundMonochorionic twin pregnancies are at increased risk of complications due to sharing a single placenta and potentially developing unbalanced vascular anastomoses. Complications include twin-twin transfusion syndrome (TTTS) which affects 10–15% monochorionic twins, and if untreated has a 70–90% perinatal loss rate. We are currently unable to predict which twins will develop complications or to what severity. We have previously shown differences in angiogenic and placental growth factors in maternal blood in pregnancies complicated by TTTS compared to twin pregnancies not complicated by TTTS but matched for gestation. There is also evidence to suggest that abnormal ultrasound measurements recorded in the first trimester (nuchal translucency and crown-rump length) may be associated with severe TTTS later in pregnancy, however the detection rate is only reported as 52%. We hypothesize that if these changes precede the development of the clinical syndrome, this may increase the sensitivity and specificity of detecting adverse pregnancy outcomes.MethodsThis cohort study has retrospective and prospective elements. In the retrospective cohort we will measure factors (decided based on preliminary work and a systematic review and meta-analysis) in stored maternal blood samples taken in the first-trimester, extract first-trimester ultrasound measurements and match these to pregnancy outcome. The prospective cohort will be divided into a “screening” cohort and “complicated” cohort. The screening cohort will undergo serial maternal blood sampling at 12, 16 and 20 weeks; we will extract ultrasound measurements and match to pregnancy outcome. The complicated cohort will comprise of women referred to the Fetal Medicine Centre with complications of monochorionicity. If the decision is taken to undergo fetoscopic laser ablation we will take maternal blood samples and amniotic fluid samples pre- and post-laser treatment. The same factors will be measured in the prospective cohort as informed by the retrospective study.DiscussionThis study aims to increase knowledge surrounding the pathology of complications in monochorionic twins, to aid future diagnosis and management.Trial registrationISRCTN 13114861 (retrospectively registered)

Prenatal central nervous system anomaly with skeletal dysplasia associated with a de novo interstitial tandem triplication of chromosome 14

Georgina K. Hall , Fiona L. Mackie , Helen Williams, Denise Williams, Phillip Cox, Dominic J. McMullan, Stephanie Allen and Mark D. Kilby West Midlands Regional Genetics Laboratory, Birmingham Women’s NHS Foundation Trust, Birmingham, UK; Fetal Medicine Centre, Birmingham Women’s NHS Foundation Trust, Birmingham, UK; Centre for Womens and Childrens Health, University of Birmingham, Birmingham, UK; Department of Radiology, Birmingham Children’s NHS Foundation Trust, Birmingham, UK; Department of Clinical Genetics, Birmingham Women’s NHS Foundation Trust, Birmingham, UK; Department of Perinatal Pathology, Birmingham Women’s NHS Foundation Trust, Birmingham, UK

Fetal Intracardiac Transfusions in Hydropic Fetuses with Severe Anemia

Introduction: Fetal anemia can have significant perinatal morbidity and mortality, particularly with onset prior to 20 weeks of gestation. Materials and Methods: We detail a case-cohort study (n = 8) of all women who underwent fetal in-utero, intracardiac transfusion prior to 24 weeks of gestation (7 women before 20 + 1 weeks), between March 2004 and September 2014, in a supraregional Fetal Medicine Center in the United Kingdom, comprising 2.2% of all transfusions performed during this period. All the fetuses were hydropic, with high maternal BMI, and had severe anemia as an indicator for transfusion. It was an attempt to perform intravascular transfusion when other common routes of fetal vascular access had failed. Results: There were 2 intrauterine deaths (25%), both of which were associated with in-utero transfusion and fulminant parvovirus B19 infection. The perinatal survival rate was 75% (6/8). Discussion: Fetal in-utero, intravascular transfusion by the intracardiac route may be used to correct severe early-onset anemia. It is particularly useful when technical issues of fetal size, early gestation (<20 weeks), maternal adiposity, and hydrops fetalis make umbilical cord or intrahepatic vein puncture technically difficult. Survival rates appear comparable to other series of pregnancies where in-utero transfusion is performed at early gestation.

PFM.11 Treatment of severe fetal anaemia before twenty weeks by intracardiac transfusion

Background Severe alloimmunisation or parvovirus B19 infection (HPVB19) may cause fetal hydrops and severe anaemia at an early gestational age (GA). Fetal anaemia is associated with significant mortality but may be successfully treated by in-utero transfusion (IUT). Perinatal loss when performed prior to 20 weeks is significantly increased compared with procedures at later gestation. One case series from the 1980’s describes the safe use of ‘intracardiac transfusion’. 1 Methods A retrospective case-cohort series of hydropic fetuses presenting in a Regional Fetal Medicine centre, prior to 20 weeks undergoing in-utero transfusion by intracardiac puncture between 2007–2013. Results Over a 6 year period a total of 202 in-utero transfusions were performed for fetal anaemia of which 7 (3.5%) were by intracardiac puncture (required due to accessing and transfusing through the umbilical vein. All pregnancies were complicated by fetal hydrops and presented at a median GA of 19.2 weeks (range 17.6–20). In 71.4% (5/7) the aetiology was HPVB19 infection, the remainder were secondary to rhesus disease (2/7). The median fetal haemoglobin at IUT was 3.3g% (range 1.3–6.1g%; all <5sd for GA) and the number of total IUTs performed ranged from 1–8. There were 2 IUDs (28.5%) associated with transfusion at early gestation (20–22 weeks). There were 5 live births (71.5%) at a median GA 36.1 weeks (range 33–38.1). Conclusion This small case cohort study indicates that intracardiac IUT is a feasible option to treat severely anaemic hydropic fetuses prior to 20 weeks gestation. Reference Bang, et al. BMJ. 1982;284:373–44