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Institute of Obstetricians & Gynaecologists, RCPI

Four Provinces Meeting

Junior Obstetrics & Gynaecology Society

Annual Scientific Meeting

Royal Academy of Medicine in Ireland

Dublin Maternity Hospitals Reports Meeting

Friday 25 November 2011

Royal College of Physicians of Ireland

No 6 Kildare Street, Dublin 2

Oral Presentations

1) NON-INVASIVE FETAL RHD GENOTYPING USING A ONE STEP REAL TIME PCR TECHNIQUE

LFA Wong, McGrath M, N Reidy, R Green

In 1998, Lo et al demonstrated that fetal RHD sequence could reliably be amplified from plasma of pregnant women with high sensitivity and specificity, eliminating the need for initial amniocentesis for fetal blood typing in alloummunised mothers. The availability of cell free fetal DNA from maternal blood to determine fetal rhesus status would also completely eliminate the use of routine administration of rhesus immunoglobulin to prevent sensitisation. To date, in the literature, various methods of recovery of cell free fetal DNA and detection of RhD antigen has been described.

A prospective study involving all rhesus negative mothers at booking visit between 12-20 weeks to allow the use of discard from any hematology samples taken routinely during their pregnancy duration for fetal Rhesus D testing.

To optimise and validate a one-step PCR assay for the detection of RhD antigen in rhesus negative maternal whole blood using the MagNa Pure Compact instrument and the Roche Lightcycler 2.0. Results are compared with serologically determined RhD status of the foetal cord blood post delivery.

63 women consented for the study. Our genotyping protocol correctly predicted the fetal genotyping in 70% (n=44) of cases. Of the remaining 30% our genotyping method failed to detect the fetal RHD gene on 27% (n=17) of antenatal women and falsely detected the RHD gene in 3% (n=2) of the women.

This initial study revealed only accuracy of 70% in the detection of fetal RHD genotype determination, highlighting the need for improvement of DNA extraction and genotyping prior to full scale introduction into clinical practice.

2) THE PPAR GAMMA AGONIST ROSIGLITAZONE REVERSES sFLT1 HYPERSECRETION FROM FIRST TRIMESTER PLACENTAL VILLI IN A GCM1 DEPENDENT MANNER

F McCarthy, S Walsh, S Drewlo, K Levytska, J Kingdom, Anu Research Centre, University College Cork, Cork University Maternity Hospital, School of Pharmacy & Life Sciences,

The Robert Gordon University, Scotland, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada

Severe pre-eclampsia is a hypertensive disorder affecting 2-3% of pregnancies and is associated with ischemic villi which increase the secretion of the anti-angiogenic protein sFLT1. Differentiation is impaired in pre-eclamptic placentas, characterized by reduced expression of the transcription factor GCM1. Peroxisome proliferator activated receptors (PPARs) are ligand activated transcription factors expressed in trophoblasts, which regulate cell differentiation and proliferation.

First trimester villous explants [8-12weeks gestation] were cultivated for 48hours in hypoxic [3%] or physiological [8%] pO2 and exposed to either: vehicle [0.5% DMSO, v/v], 1-100µM Rosiglitazone, a PPAR-ã agonist or 0.05-0.5µM T0070907, a PPAR-ã antagonist. PPAR-ã activation was assessed by CHIP assay. RNA was extracted to monitor GCM1 mRNA using qRTPCR. In parallel, GCM1 was silenced using siRNA. ELISAs were used to measure HO-1 and sFLT1 expression.

The molecular mechanisms causing increased production of sFLT1 are unknown. We tested the hypothesis that normal placental differentiation represses sFLT1 via the GCM1 axis and this process is regulated by PPAR-ã.

PPAR-ã activation significantly increased binding to target DNA [+23.5%vs.,n=4] and mRNA expression of GCM1 [5.9±1fold,n=7]. These changes reduced sFLT1 [59.7±9.8%,n=9] and increased HO-1 (a cytoprotective enzyme) secretion, [57.9%±27,n=9]. In contrast, T0070907 reversed all these observations. GCM1 knock- down significantly induced sFLT1 secretion [59.2%±13] and proved GCM1 dependency (All data P ................
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