Prenatal Screening, Fetal Medicine & Therapy

(A/Prof Mahesh Choolani, A/Prof Citra Mattar)

Prenatal Screening

Cell-based

Circulating fetal cells are rare and a promising source for non-invasive detection of aneuploidies as well as for providing complete genetic information of the fetus. Through our research, we have developed several novel strategies, which includes biomarkers and microfluidic devices that could consistently enrich fetal nucleated red blood cells (FNRBCs) from maternal blood. We also showed the feasibility of DNA whole genome amplification from a single FNRBC, followed by massively parallel sequencing, to detect chromosomal abnormalities such as trisomies 21, 18, and 15. Correct diagnoses were made from single cells isolated from villi from two cases of trisomy 21, two cases of trisomy 18, and a case of trisomy 15. This shows potential utility for definitive non-invasive prenatal diagnosis.

Cell-free fetal DNA

Cell-free fetal DNA (cffDNA) that originates from the placenta and circulates in maternal blood is detectable from five weeks' gestation onwards, constitutes 10-15% of a cfDNA sample, the remainder being maternal in origin. High maternal background makes it difficult to identify maternally inherited fetal alleles. Our solution was to use targeted next generation sequencing panel of 35 insertion/deletion (INDELS) polymorphisms for universal quantification of cffDNA fractions.

Sequencing four most common South East Asian β-thalassaemia mutations and HbE, we achieved reliable detection of paternally inherited alleles with 100% sensitivity. Therefore, removing the need for an invasive test in 2/3 of patients referred for prenatal testing of paternally inherited β-thalassaemia

Alloimmunisation against the D-antigen in RhD-negative pregnant women is preventable using anti-D immunoprophylaxis and dramatically decreases the risk of haemolytic disease of the fetus and newborn. Analysis of cffDNA facilitates non-invasive prenatal testing of the fetal RhD genotype during pregnancy. International collaboration in an external quality assessment exercise resulted in a robust and reliable laboratory RhD-genotyping assay, which may be used as a guide for targeted Rh prophylaxis and avoid unnecessary treatment of RhD-negative women carrying an RhD-negative fetus.

Fetal Medicine and Therapy

Prenatal and Early Neonatal Gene Therapy Strategies using a Clinically Relevant Lentiviral Vector in a High-Fidelity Mouse Model to address Critical Translational Questions

β-thalassaemia major, a global disease causes 3-4% of early-childhood deaths annually, with severe chronic morbidity arising from progressive disease, inadequate treatment and lack of curative haemopoietic stem cell transplantation (HSCT). To avoid the immune and physical barriers that complicate post-natal therapy, treatment is instigated during early fetal development. We investigate (i) intrauterine gene therapy (IUGT), and (ii) intrauterine HSC transplantation (IUHSCT) in a murine fetal model of severe thalassaemia whereby heterozygote offspring share phenotypic similarities with β-thalassaemia intermedia while homozygote offspring represent α-thalassaemia major. A single intrahepatic injection of 3rd generation lentiviral (LV) GLOBE2.0 vector expressing the human β-globin transgene efficiently corrects the severe phenotype of the HbbTh3/+ β-thalassaemia mouse when administered perinatally is compared to ex-vivo GT. We aim to assess GLOBE2.0 transduction efficacy, compare phenotype correction and interrogate long-term biosafety. The fetal medicine and therapy research group has a long-standing collaboration with Prof Jerry Chan at KKWCH and international collaborators to investigate important clinical outcomes of intrauterine gene and stem cell therapy in non-human primate models.

Mouse models allow for understanding of the interactions between maternal and fetal immune systems and strategies to be developed to improve donor cell engraftment. Several strategies are investigated for the improvement of long-term engraftment such as interrogating the effects of a transient depletion on maternal antigen-presenting cells on maternal immune tolerance induction, donor cell chimerism and long-term engraftment in transplanted offsprings. In addition, ex vivo priming of donor HSCs to express cytokines and chemokines associated with homing, migration and adhesion to bone marrow endothelial cells by co-culture systems with amniotic fluid stem cells may improve long-term engraftment within the haemopoietic niches.

Amniotic Fluid Stem Cell Conditioned Medium for Vascular Tissue Regeneration in a Murine model of Ischemic Heart and Peripheral Artery Disease

Amniotic fluid stem cells (AFSC) posses beneficial cellular properties such as higher proliferation and differentiation compared to adult-derived stem cells. Furthermore, human AFSC possess high self-renewal potential similar to embryonic stem cells but without the concerns of tumorigenicity and ethical issues. The AFSC secretome (AFSC-S) analysis has shown to contain pro-angiogenic factors and cytokines which promote tissue repair. Application includes the use of the secretome alone to prime various cell types involved in tissue healing and repair to enhance recovery after organ injury, such as cardiomyocytes and vascular cells in cardiovascular diseases (CVD). Utilising AFSC-S we aim to evaluate the mechano-transduction signalling pathways of human cardiomyocytes and endothelial cells cultured in normoxic and hypoxic conditions under the influence of human AFSC-CM, evaluate the potential therapeutic application of AFSC-CM in reversing tissue injury in central and peripheral vasculature ischemia animal models; and finally to optimize the lyophilisation of AFSC-CM for in vivo applications.

Fluid Mechanics of Fetal Cardiovascular System

Congenital structural heart diseases affect 0.6-1.9% of the population, and results in considerable mortality and morbidity despite advances in surgical therapy. Recent investigations showed that the mechanical force environment in the fetal circulation affects the structural development of the cardiovascular system, leading to the hypothesis that adverse mechanical environment play a role in causing congenital structural cardiovascular diseases. Thus, it is important that we improve our understanding of the mechanical force environment of the fetal circulation by characterising the fluid mechanical force environments in fetuses with normal and malformed cardiovascular systems (using echocardiographic, CT, MRI, or ultrasound scans together with engineering computations), of fetuses before and after birth to understand how the blood flow forces influence the growth and remodelling of the fetus’ cardiovascular anatomy. We envision that in the near future, all congenital cardiovascular malformations in fetuses will be treated through surgery before birth (fetal surgery), rather than after birth, such that we can tap into the regenerative ability of the fetus to obtain improved outcomes.

Shear Wave Imaging of the Placenta Studies

Intrauterine Growth Restriction (IUGR) has a prevalence of 3% in the developed world and 10-15% elsewhere. The underlying cause of IUGR is placental insufficiency. The IUGR fetus has a 5- to 10- fold increase in mortality risk and increased risk of impaired development of renal- and neuro-systems after birth. The fetus is also predisposed to adult onset hypertension, type II diabetes, and cardiovascular diseases. There is currently no proven strategy to prevent or treat IUGR, but studies have shown that early detection improves outcome. Current techniques such as echocardiography biometric measurements of fetal dimensions, umbilical vessel Doppler flow measurements, and amniotic fluid volume estimation for detection of IUGR suffer from low sensitivities. We utilise Shear Wave Imaging modality to discriminate, study the micro-structural and -mechanical property differences between normal and IUGR placentae. We hypothesise that SWI is an accurate method for detecting IUGR and perform clinical validation.

On-site and tele-mentoring of a novice surgical team towards procedural competence in the early learning curve of fetoscopic surgery: a model for cross-border fetal therapy services

Fetoscopic laser coagulation of arterio-venous anastomoses (AVA) in a monochorionic placenta is the standard of care for twin-twin transfusion syndrome (TTTS), but is technically challenging and can lead to significant complications. Selective fetoscopic laser photocoagulation (SFLP) for twin-to-twin transfusion syndrome (TTTS) carries a long learning curve. New proceduralists may have neither the resources for traditional apprenticeship nor the caseload to meet numeric requirements for operator proficiency. Training on realistic surgical simulators can potentially shorten this steep learning curve and enables several proceduralists to acquire procedure-specific skills simultaneously. We have studied and published on alternative approaches such as the usage of realistic simulators designed to allow the user familiarity with the equipment and specific steps required in the surgical treatment of TTTS, including fetoscopic handling, approaches to anterior and posterior placenta, recognition of anastomoses, and efficient coagulation of vessels. These models can be adapted easily depending on the availability of materials and require standard fetoscopy equipment. We concluded that such training systems are complementary to traditional surgical apprenticeships and can be useful aids for fetal medicine units that provide this clinical service.

Clinical Translation

Rapid prenatal diagnosis

Rapid aneuploidy detection assay using quantitative fluorescence polymerase chain reaction of 19 microsatellite markers located on chromosomes 13, 18, 21, X and Y, provides results within 48-hours and was validated in over one thousand samples. FlashFISHTM, a test developed in 2008, tags chromosomes 13, 18, 21, X, and Y in each fetal cell with their respective fluorescent-labeled probes, which enables visualisation and numeration of chromosomes under a fluorescence-enabled microscope.

Pioneer in in utero fetal stem cell treatment in Asia

NUH played a key role in the first fetal stem cell treatment in Asia. International collaboration with researchers from Singapore, Taiwan and Sweden made it possible; to date we have treated two fetuses in utero diagnosed with ostegenesis imperfecta. Both recipients are experiencing normal growth trajectories and their long-term clinical course is being monitored.