Research

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Scientific Session

 

 

 

Prevalence and predictors of folic acid use during pregnancy in a large Irish obstetric cohort

McGuire M, Cleary B, Sahm L, Murphy DJ
Department of Pharmacy and Department of Obstetrics & Gynaecology, The Coombe Women and Infants University Hospital, and Trinity College Dublin.

Introduction: Ireland has one of the highest incidences of Neural Tube Defects (NTDs) in Europe, affecting
approximately 1-1.5 per 1,000 births nationally. Neural tube defects (NTDs) are severe abnormalities of the central nervous system that develop in babies during the first weeks of pregnancy. Evidence has led to the belief that deficiency FA is a cause of NTDs. FA may exert its action through correction of a metabolic defect.

Objectives:The objectives of this study were to determine the prevalence and socio-economic predictors of folic acid use during pregnancy in a large obstetric cohort.

Setting: This descriptive study was carried out on the medical records database of the Coombe Women and Infants University Hospital, Dublin covering 2000-2007.

Method: A descriptive cohort study was carried out on the medical records database of the Coombe Women and Infants University Hospital, Dublin covering 2000-2007. Data was analysed using Statistical Package for the Social Sciences Version 15 (SPSS®).

Key findings:

• 85% of women reported taking FA at some time.
• 27.7% of women reported taking FA optimally.
• Being, Irish, of higher SES, nulliparous, married, of older age and having a planned pregnancy was associated with
  higher reported FA uptake and optimal use.
• Smoking, illicit drug and higher alcohol use and social worker referral were associated with lower FA use and optimal use.
• Patients receiving fertility treatment had the highest reported optimal FA use.
  Conclusion: Socio-Economic factors do influence use of FA. Vulnerable groups such as the unemployed, women less than 20 years of age and women with unplanned pregnancies should be specifically targeted in future Public health Campaigns. A multi-pronged approach involving fortification, taking FA supplements and eating a diet rich in FA and a multimedia campaign may be more successful in improving FA uptake in women of child bearing age.

 

Are Fetal Abdominal Subcutaneous Tissue (FAST) measurements using antenatal ultrasound different between male and female fetuses?

E Harrold, N Farah, C Fattah, S Barry, V Donnelly*, G Rafferty*, B Stuart, MJ Turner.
UCD School of Medicine and Medical Sciences, Coombe Women and Infants University Hospital, Mount Carmel Hospital*.

Abnormal fetal growth increases the complications of pregnancy not only for the baby but also for the mother.
Despite the use of more than 50 different formulae to determine EFW, ultrasound has its limitations especially at the extremes of fetal weight. There is emerging interest in fetal body composition. Babies born to diabetic mothers or born growth-restricted have abnormal adiposity. After birth, the pattern and distribution of subcutaneous fat has been assessed by various methods and gender differences have been reported. The purpose of this study was to determine if the FAST measurements using antenatal ultrasound differ between the sexes.

Women who had an ultrasound examination for fetal growth between 20 and 40 weeks gestation were analysed. Women with diabetes mellitus were excluded. The fetal anterior abdominal subcutaneous tissue was measured in a standardised way. The fetal sex was recorded after delivery (EH).

A total of 557 fetuses were measured, 290 male and 267 female. The FAST increased at the same rate for both male and female fetuses and at any given week there was no gender difference. The increased fat composition in females reported after birth was not found in abdominal wall subcutaneous fat measurements using ultrasound during pregnancy. Antenatal centile charts for FAST do not need to be based on gender.

 

Complications related to chorionicity in twin pregnancies.

N Farah, J Hogan, B Stuart, S Daly.
Coombe Women and Infants University Hospital.

Twin pregnancies are associated with higher complication rates when compared to singletons. They are mainly at an increased risk from preterm delivery and perinatal death. We sought to investigate these risks and comparing them between monochorionic and dichorionic pregnancies.

We analysed our twin database from July 1999 till July 2007 and included all twins where one twin weighed more than 500g or was over 24 week’s gestation. Chorionicity was assigned using information on the sex of the babies, the ultrasound findings and the pathology reports.

There were 972 twin pairs delivered, that met the inclusion criteria, of which 740 (76%) were dichorionic, 221 (22.7%) monochorionic and in 11 twin pairs we were not able to assign chorionicity. Table 1 outlines the risk of preterm delivery for both monochorionic and dichorionic twins. The risk of perinatal death was 9.7% (43) for monochorionic twins while 2.8% (42) for dichorionic.

Monochorionic twins are more likely to result in perinatal death and preterm delivery. The data presented is useful in counselling patients with twin pregnancies.

Gestation (weeks)	Monochorionic %	Dichorionic %
By 26	3.6 (n 8)	2 (n 15)
By 28	9 (n 20)	2.3 (n 17)
By 32	17.2 (n 38)	6.6 n 49)
By 36	39.4 (n 87)	27.4 (n 203)
By 38	74.7 (n 165)	63.9 (n 473)
By 40	100 (n 221)	99 (n 733)

 

TLR-9 senses human fetal DNA.

Andrea S. Nugent*#, Sean Daly*, John O’Leary* and Luke AJ O’Neill# PhD
#School of Biochemistry and Immunology, Trinity College Dublin.
*The Coombe Women and Infants University Hospital, Dublin, Ireland.

Toll-like receptor 9 (TLR9) senses CpG motifs in DNA. These are more common in bacterial and viral DNA and TLR9 has been shown to have an important role in the sensing of various pathogens during host defence. Recent studies have suggested that abnormal epigenetic changes in CpG-rich islands in fetal mammalian DNA might contribute to the high rate of early pregnancy loss. It is also well known that higher concentrations of free fetal DNA are found in mothers who deliver prematurely. We therefore wished to test the hypothesis that fetal DNA might be sensed by TLR9, and might provoke an inflammatory response, which in turn could lead to preterm labour and early pregnancy loss.

Our investigations have shown that fetal DNA added to the Namalwa B cell line (which expresses high levels of TLR9) or PMBCs could rapidly activate NF-kappaB (as measured by increased I-kappaB degradation) and also p38 activation, both of which are typical TLR9 signals. We also found increased production of the pro-inflammatory cytokines IL6 and TNF. The effects of fetal DNA were more potent that either synthetic CpG – containing oligonucleotides, or adult DNA. We also found that chloroquin, which has been shown to inhibit TLR9 signaling, blocked the effect. Finally, we have found that the effect of fetal DNA on cytokine induction is significantly reduced in TLR9-deficient bone marrowderived macrophages. We have therefore made the novel observation that TLR-9 senses fetal DNA and facilitates an inflammatory reaction. This may then contribute to preterm labour or early pregnancy loss.

 

Real-time PCR analysis for the rapid early detection of Pseudomonas aeruginosa in paediatric Cystic Fibrosis patients.

Logan, Catriona1, Lennon, Grainne1,2, Habbington, Adele1, O’Leary John J.1,3 and O’Sullivan Niamh1,3.

1. Molecular Microbiology Department, Our Lady’s Children’s Hospital, Crumlin, Dublin 12.
2. The Children’s Medical and Research Foundation, Our Lady’s Children’s Hospital, Crumlin, Dublin 12.
3. Pathology Department, Coombe Women and Infants University Hospital, Dolphins Barn, Dublin 8.

Introduction: Recurrent chronic chest infections caused by Pseudomonas aeruginosa are a common complication of Cystic fibrosis (CF). Initial P. aeruginosa infections are intermittent and sensitive to anti-microbials. However mutations in Pseudomonas mucA occur during infection, results in conversion to a ‘mucoid’ phenotype. Mucoid P. aeruginosa cannot be eradicated with antibiotics, resulting in permanent chronic colonization of CF lungs. Conversion to mucoidy can occur as early as 3-months from initial infection. Epidemic spread of P. aeruginosa isolates among CF patients has been reported. Prompt diagnosis and early antibiotic treatment are essential, as P. aeruginosa eradication is only likely prior to conversion mucoidy.

Methods: Real-time PCR assays for P. aeruginosa detection were designed, optimised and validated for the analysis of total nucleic acids extracted from respiratory specimens. More than 2000 respiratory specimens from 175 CF patients received for molecular analysis over an 18-month period were analysed by PCR and results compared with conventional microbiological sputum culture results.

Results: Real-time PCR analysis results in increased detection of P. aeruginosa in CF respiratory specimens as compared to conventional microbiological culture.

The UK Manchester and Liverpool epidemic P. aeruginosa strains were not detected in the patient cohort.

Real-time PCR highlights the inadequacy of throat swabs for the determination of P. aeruginosa colonisation status. Analyses identified >5% of specimens as “insufficient for PCR analysis” based on criteria defined for the maximum Ct value for human beta-actin DNA detection. Specimen type was the predominant factor in relation to the observance of specimens “insufficient for PCR analysis” with 14.0% of throat swabs and 6.1% of sputa deemed insufficient. Of specimens deemed “insufficient for PCR analysis”, >95% were reported culture negative for P. aeruginosa.

 

Prevalence and Genotype Distribution of Human Papillomavirus in the Irish Cervical Screening Population.

Mc Inerney J1,2, Pilkington L1, Keegan H1, Ring M1, Bolger N1, Casey M3, Sheils O2, O’Leary JJ 1,2, Martin CM 1,2

1. Department of Pathology, The Coombe Women and Infants University Hospital, Dublin 8, Ireland.
2. Department of Histopathology, University of Dublin, Trinity College, Dublin 2, Ireland.
3. Department of Pathology, University College Hospital Galway, Ireland.

Objectives: Human Papillomavirus (HPV) infection is the causative agent in the development of cervical cancer. HPV types 16 and 18 are detected in greater than 70% of cervical carcinomas. Two vaccines, Cervarix™ and Gardasil®, have recently come on the market targeting these high risk types. With the introduction of a national HPV vaccination program, it is essential that HPV genotype prevalence in the Irish screening population is determined. The aim of this study is to determine prevalence and genotype distribution of HPV in the Irish screening population. This study forms part of the CERVIVA program funded by the Health Research Board, Ireland.

Methods: To date, 1229 Thin Prep® cervical specimens from women attending GP clinics for routine cervical smear tests were recruited to the study through the Department of Cytology, Coombe Women & Infants University Hospital, Dublin and University College Hospital, Galway. Samples recruited to these centres are representative of the screening population in Leinster and Connaught. Cytological diagnosis was made according to BSCC guidelines. High risk HPV DNA was detected using Hybrid Capture II assay (hc2, Digene Ltd., UK). hc2 detects 13 different high risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) in cervical specimens. Samples positive for HPV DNA were genotyped for 37 HPV types using the Roche Linear Array HPV Genotyping Test.

Conclusions: The prevalence of HPV in this screening population was 18%. Abnormal cytology was present in 10% of samples in this population. HPV16 (31.5%) was the most common type detected, followed by HPV39 (13%), HPV18 (12%), HPV66 (12%) and HPV 31(10.6%). The presence of high risk types other than 16 and 18 highlights the importance of continued cervical screening in a HPV vaccinated population. Further studies are underway to determine prevalence throughout Ireland.

 

Human Papillomavirus Prevalence and Persistence, in the Cervix of Women with Human Immunodeficiency Virus.

Martin C.M.1, Mc Inerney J1, Keegan H1, Pilkington, L1, C. Ni Cheallaigh2, S. Delamere2, M. Griffin3, G. McHugh2, P. Webster3, M. Barrett3, F. Lyons2, C. Bergin2, F. Mulcahy2, S. Clarke2, Sheils O1, O’Leary JJ 1

1. Department of Histopathology, University of Dublin, Trinity College, Dublin 2, Ireland & The Coombe Women and Infants University Hospital.
2. Department of Genitourinary Medicine and Infectious Diseases, St James’s Hospital, Dublin 8, Ireland.
3. Department of Cytology, St James’s Hospital, Dublin 8, Ireland.

Objectives: HPV is the major aetiological factor in the development of cervical intraepithelial neoplasia (CIN) and cervical cancer. Human immunodeficiency virus (HIV) positive patients are at greater risk for CIN and persistent HPV infections. In this study we examine the prevalence and persistence of HPV in cervical specimens from women with HIV and the effect of Antiviral Retro Therapy (ART) on HPV status and cervical abnormalities. This study forms part of the CERVIVA program funded by the Health Research Board, Ireland

Methods: PreservCyt™ cervical smears from 150 HIV positive women were prospectively recruited through the
Genitourinary and Infectious Disease Clinic at St James’s Hospital Dublin. Samples are taken at time of recruitment and annually where patient is stablised on ART or 3 monthly where patient has commenced ART. CD4 counts and HIV viral load were determined for each patient at baseline. Cytological diagnosis was made according to BSCC guidelines. High risk HPV DNA status was assessed using Hybrid Capture 2 (Digene) (hc2). HPV E6/E7 mRNA expression was detected using PreTect HPV Proofer (Norchip) which detects HPV 16, 18, 31, 33, and 45.

Results: 29% (44/150) of HIV positive women examined in this study had abnormal cytology. The HPV prevalence rate in this population of HIV positive women was 50% (75/150), while the prevalence of HPV E6/E7 mRNA expression in this population was 19% (28/150). Among the women with cytological abnormalities 86% were positive for high risk HPV DNA with 39% positive for HPV E6/E7 mRNA. HPV 45 (11/28) was the most predominant genotype followed by HPV 33 (9/28), 16 (8/28), 18 (7/28) and 31 (2/28). The rate of multiple HPV type infections was 32%. In women with follow up smears on ART, HPV persistence rates for HPV DNA and RNA were 32% and 21% respectively. The rate of HPV positivity correlated with low CD4 counts (<200 x 10 6/L), while no correlation was observed with HIV viral load.

 

Reduction of severity of pruritus after elective caesarean section under spinal anaesthesia with intra-thecal morphine: A comparison of prophylactic administration of Granisetron with Ondansetron.

T. Tan, R. Ojo, S. Immani and M. Carey.
Department of Peri-operative Medicine, The Coombe Women and Infants University Hospital.

The incidence of pruritus in parturients after elective caesarean section under spinal anaesthesia with intrathecal morphine has been reported to be between 60% to 100%, and is a common cause of maternal dissatisfaction. Ondansetron has been shown to reduce pruritus but the effect is short-lived. The objective of this randomised controlled double-blind trial was to evaluate the anti-pruritic efficacy of Granisetron compared with Ondansetron. Eighty ASA I or II women undergoing elective caeserean section under spinal anaesthesia were randomised to one of two groups. Spinal anaesthesia was performed in both groups with 10mg of 0.5% hyperbaric bupivacaine, Fentanyl 25μg plus 150µg preservative free morphine administered intra-thecally. After delivery of the baby and umbilical cord clamped, one group (G) received granisetron 3mg IV; the seocnd group (O) received ondansetron 8mg IV at the equivalent time.

The two groups were similar in terms of age, gestational age, height and weight. According to visual analogue pruritus scores, patients in group G experienced less pruritus at 8 hours (P=0.003) and at 24 hours (P=0.01). Fewer patients in group G (8) required rescue anti-pruritic medication than group O (18) (P=0.03). There was also higher satisfaction scores in group G compared to group O (P=0.0252). There was no difference in the overall incidence of pruritus, nausea and vomiting, and visual analogue pain scores between both groups of patients.

Administration of Granisetron 3mg IV markedly reduces pruritus, use of rescue anti-pruritic medication, and improves satisfaction but does not reduce the overall incidence of pruritus when compared to Ondansetron 8mg IV.

 

Prescribed and Non-prescribed Medication Use in Early Pregnancy in a Prospective Cohort of Women

Brian J Cleary, Hajeera Butt, Judith Strawbridge, Paul Gallagher, Tom Fahey, Deirdre J Murphy.
Department of Pharmacy and Department of Obstetrics & Gynaecology, The Coombe Women and Infants University Hospital, and Trinity College Dublin.

Objectives: To determine the extent and nature of medication use in early pregnancy, exploring inappropriate
prescribing with potential for fetal harm and prescribing for known medical disorders.

Design: Descriptive study.

Setting: Tertiary referral obstetric hospital in Dublin, Ireland.

Participants: All women who booked for antenatal care and had a corresponding delivery record between 2000 and 2007.

Main outcome measures: Prevalence and determinants of medication use for any indication, inappropriate prescribing with potential for fetal harm referenced against FDA pregnancy categories and appropriateness of prescribing for chronic medical disorders referenced against clinical practice guidelines.

Results: Excluding folic acid, use of at least one medication was reported in 23989 (39.2%) pregnancies. 11970 (19.5%), 545 (0.9%) and 352 (0.4%) women reported taking OTC medications, illicit drugs (including methadone) and herbal medicines / supplements respectively. FDA category D and X medications were reported by 1913 (3.1%) and 1987 (3.2%) women. Factors associated with reporting use of any medication included being over 35 years of age, having an unplanned pregnancy, being single, smoking during pregnancy, drinking alcohol prior to pregnancy and being a private patient. Medications with potential for fetal harm were reported more often among those with unplanned pregnancies, the unemployed, single women, smokers and publicly-funded patients. Asthma, depression and hypertension were among the most commonly reported chronic medical disorders. Medications known to cause fetal harm were reported by 86 (15.7%) women treated for depression and 68 (20%) women treated for hypertension.

Conclusions: A significant proportion of women report using medications before booking for antenatal care. Women and prescribers need to be aware of the lack of safety data for many medications that are frequently taken during pregnancy and the need for pre-pregnancy planning. Prescribers should exercise caution when prescribing medications with potential to cause fetal harm to women of childbearing potential.

 

POSTER PRESENTATIONS

 

 

 

ECSSIT – Elective Caesarean Section Syntocinon® Infusion Trial: The first 100 patients.

Sharon Sheehan, Michael Carey, Deirdre Murphy, on behalf of the ECSSIT Group.

Objective: The aim of ECSSIT is to compare the blood loss at elective lower segment caesarean section with
administration of oxytocin 5IU bolus versus oxytocin 5IU bolus and oxytocin 40IU infusion. We will analyse the first 100 patients recruited at the Coombe Women and Infants University Hospital to this trial. The baseline characteristics, operative factors and peri-operative outcomes of these patients will be assessed, blinded.

Study design: Women booked for elective caesarean section are recruited to the randomised controlled trial and randomised to receive either oxytocin bolus and placebo infusion or oxytocin bolus and oxytocin 40 IU infusion. The outcome measures are measured blood loss at caesarean section and the need for an additional uterotonic agent.

Results: Of the first 100 women recruited, 70% had a previous caesarean section. The total mean measured blood loss was 822mls. 27 women had a major haemorrhage (measured blood loss >1000ml). An additional uterotonic agent was required in 15 women. 18 women had pre-op anaemia (Hb<10.5d/dl), whilst 55 women had post-operative anaemia. The trial infusion was used in 96 women and discontinued in 12.

Conclusion: It is interesting to see the high incidence of major obstetric haemorrhage (27%) among this population. We eagerly await the conclusion of the trial and to see the results in each arm.

The study aims to recruit 2000 women, 1000 in each arm. To date (beginning September 2008), 400 have been recruited across the 3 Dublin maternity hospitals. The target end date is Summer 2010.

 

Caesarean section surgical-site infection: incidence and risk factors

Authors: Adeeb Khalifeh, Anne-Marie Meenan*, Rosena Hannify*, Niamh O’Sullivan*, Chris Fitzpatrick, Michael J Turner UCD School of Medicine and Medical Science & the Microbiology Department*, Coombe Women and Infants University Hospital, Dublin 8, Ireland.

Objective: To determine the incidence of wound infection and independent risk factors following caesarean section. Patients and Methods: A prospective audit was conducted on 99 women who delivered by caesarean section in July- August 2008. All women received prophylactic intravenous cefuroxime (1.5 g intravenously) given at cord clamping. All wounds, belonging to the clean-contaminated class, were reviewed before discharge. Their microbiological results were also reviewed if they presented to the hospital after discharge. Different clinical parameters were recorded using a protocol adapted from Northern Ireland on caesarean section surveillance (See Table 1). The definition of surgical site infection was standardised.

Results: The overall wound infection incidence was 7% (7 patients); 5 were diagnosed as inpatients and 2 as
outpatients. Three were elective caesarean sections and four were emergency caesarean sections, one of which not in labour. All sections lasted less than one hour. There were no independent risk factors identified, including Diabetes Mellitus and immunosuppression. The organisms isolated are shown in Table 2. Wound infection did not prolong length of stay as inpatients.

Conclusion: The incidence of 7% is similar to previous studies elsewhere. The datasheet used in our analysis will be rolled out nationally by the Health Protection Surveillance Centre (HSE) as part of the strategy for the control of Antimicrobial Resistance in Ireland (SARI). The next step is to assess the timing of prophylactic antibiotics on a larger number of patients; would that influence the incidence of infection after caesarean section?

	BMI (Average)	Membrane rupture	Previous CS	Diabetes Mellitus	Immunosuppression
Total n=99	25.3kg/m2	4.6hrs	38	3	3
Infected wounds n=7	26.0 kg/m2	2.6hrs	1	1	0

 

Organisms	Number of cases
Staphylococcus Aureus	3
E Coli	2
Mixed Anaerobes	4
Enterococcus faecalis	3
Proteus Mirabilis	1
Group B Streptococcus	1

 

Vaginal delivery for the second twin, is it safe?

N Farah, J Hogan, B Stuart, S Daly.

Coombe Women and Infants University Hospital.
The mode of delivery in twins is controversial. Some institutions suggest that all twin pregnancies are delivered by caesarean section as there is significant morbidity in delivering the second twin by caesarean section. We analysed our twin database from July 1999 till July 2007 and included all twins where one twin weighed more than 500g or over 24 weeks gestation. There were 972 sets of twins identified that met the criteria, in 11 sets the chorionicity could not be assigned and these were not included in the analysis.
In 509 sets both twins delivered vaginally, in 17 cases the first twin delivered vaginally while the 2nd twin was
delivered by an emergency caesarean section. Of those delivered by emergency caesarean section there was only one case were the apgar score at 5 minutes was less than 7. In 8 cases twin 2 was heavier than twin 1, and in 13 cases the consultant was present at the delivery.

Vaginal delivery should be anticipated in twin pregnancies as in our population it occurred in 53%. The need to
perform an emergency caesarean section for the second twin is rare (3.2%). If the second twin is delivered by
caesarean section, this is not associated with any significant morbidity.

 

Does the Placenta help to identify Identical Twins?

J Hogan, N Farah, Z Galvin, A Radomski, J O’Leary, B Stuart, S Daly.
Coombe Women and Infants University Hospital, Dublin.

The placenta from twin pregnancies are routinely sent for histopathology in some institutions. Pathological
assessment to determine chorionicity is considered by many to be the gold standard. We sought to investigate if this is actually the case.

A retrospective analysis of our hospital’s database of all twins (delivered at or after 24 weeks gestation or weighing equal to or greater than 500g) from July 1999 to July 2007 was undertaken. The sex of the infants, the placental pathology and the ultrasound reports were reviewed.

Using the above criteria, there were 972 twin pairs. Of these, 806 pairs had the placenta sent for histological
examination (82.9%). Table 1 displays the classification of chorionicity by pathology. There were 198 classified
as monochorionic but 16 of these had different sex infants. This means that 8% had been wrongly classified as monochorionic when they were in fact divhorionic twins. In 12 of these cases, ultrasound concurred with the fetal sex and these twin pairs had been classified as dichorionic during their pregnancy.

Our study suggests that the placental pathological assessment of chorionicity is accurate in most but not all cases. If these results are replicated in other institutions, radiological plus histological determination of chorionicity may become the gold standard in the future.

Chorionicity	n
Dichorionic	583
Monochorionic	198
Unknown	25

 

Ultrasound Placental Localisation After One Previous Caesarean Section

J Hogan, N Farah, B Stuart, MJ Turner
UCD School of Medicine and Medical Science, Coombe Women and Infants University Hospital, Dublin.
A prior lower segment caesarean section has been reported as a risk factor for placenta praevia. Women with a prior caesarean section are more likely to require repeat section and abnormal placental localisation makes it technically more challenging. To our knowledge, there are no studies reported which identify the placental site in women with one prior caesarean section.

We reviewed women with one prior caesarean section who delivered over a four month period and who had the placenta localised during a formal ultrasound in the second or third trimester. The smoking history, previous uterine curettage and parity was also recorded.

Of 262 patients delivered with one prior caesarean section, 170 had a departmental ultrasound. Three patients had placenta praevia and there was no case of placenta accreta. Table 1 shows the placental site distribution. We found no association between placental site and either parity or previous uterine curettage. The three patients with abnormal placental sites all had a smoking history but the numbers are too small to be conclusive.

Previous ultrasound studies have focused solely on abnormal placentation. Our study highlights that while the
incidence of placenta praevia after caesarean section is low (1.8 %), it is advantageous for the obstetrician to know the site of the placenta preoperatively to plan the uterine incision.

Table 1. Placental localisation on antenatal ultrasound

Placental site	n (%)
Placenta praevia	3 ( 1.8)
Upper segment anterior	75 (44.0)
Upper segment posterior	65 (38.2)
Upper segment lateral	18 (10.6)

 

Syphilis- A Review Of Management & Outcome In A Tertiary Referral Unit

Dr. Meenakshi Ramphul, Orla Phelan, Dr Michael O’Connell.
The Coombe Women & Infants University Hospital.

Introduction: The emergence of syphilis in recent years has posed many problems in the antenatal period.

Aim: This study aims to identify management and outcome of those seen in the antenatal period with positive
serology in the Coombe Women & Infants Hospital.

Methods: Data was collected retrospectively from 2005 to 2007 and prospectively in 2008. A review of antenatal and paediatric charts was undertaken.

Results: A total of 39 cases were identified, 18 of which were of known history of syphilis. A total of 20 women
underwent treatment in the pregnancy. Of the 39 cases, 19 were of Eastern European origin, 12 African, 1 South American, 4 Asian with only 2 of Irish origin. All cases identified were reviewed by the GUIDE Clinic and all babies were referred to the Rainbow Clinic. There was no adverse perinatal outcome in the cohort.

Conclusion: Syphilis is an emerging albeit rare antenatal management issue. This study illustrated the value of a multidisciplinary approach to the study.

 

Molecular typing of Pseudomonas aeruginosa isolates from paediatric Cystic Fibrosis patients.

Logan, Catriona1, Habbington, Adele1, Lennon, Grainne1,2, O’Leary, John J.1,3 and O’Sullivan Niamh1,3.

1. Molecular Microbiology Department, Our Lady’s Children’s Hospital, Crumlin, Dublin 12.
2. The Children’s Medical and Research Foundation, Our Lady’s Children’s Hospital, Crumlin, Dublin 12.
3. Pathology Department, Coombe Women and Infants University Hospital, Dolphins Barn, Dublin 8.

Introduction: While cystic fibrosis is a multi-organ disease, respiratory infections, particularly chronic chest infections caused by the opportunistic pathogen Pseudomonas aeruginosa, represent a major cause of morbidity and mortality. We are undertaking a three-year study focusing on P. aeruginosa detection and characterisation. Early detection of P. aeruginosa provides the opportunity to eradicate infection before development of the chronic mucoid phenotype. Characterisation of isolates by molecular typing enables the identification of predominant, possibly transmissible, strains of P. aeruginosa in the Irish CF population.

Highly transmissible strains of P. aeruginosa, some of which show increased anti-microbial resistance, have been identified in a number of countries and constitute an emerging threat to CF patients. Reports suggest that CF patients infected with transmissible strains of P. aeruginosa show increased morbidity and mortality. It is not currently known if transmissible strains of P. aeruginosa exist in the Irish CF population and the identification of possible transmissible strains within the Irish CF population is a fundamental prerequisite to optimise patient care.

Methods: Molecular typing of >300 P. aeruginosa isolates from 46 CF patients was performed by both Pulsed Field Gel Electrophoresis (PFGE) and Fluorescent-Amplified-Fragment-Length-Polymorphism (FAFLP), a method capable of identifying micro-heterogeneity among similar bacterial strains. Fingerprinting II software (Biorad) was used for pyhlogentetic analyses of genotype patterns.

Results: Analysis of molecular typing profiles of P. aeruginosa isolates has identified that:
In all cases (9 sets of siblings) where 2 or more siblings are culture positive for P. aeruginosa, the siblings harbour isolates with the same molecular fingerprint.

• In cases of intermittent infection with P. aeruginosa and attempted eradiation, re-infection is often caused by the same bacterial strain.
• Approximately 20% of CF patients examined to date are harbouring the same P. aeruginosa strain. This has enormous implications for clinical practice and infection control. This is the first report of a clonal P. aeruginosa strain in Irish CF patients, and the possibility that this strain is transmissible and may have increased virulence requires further investigation.

Holoclone and non-holoclone derived cell lineage miRNA analysis in prostate cancer

YM Salley1, S Elbaruni, P Smyth1, CM Martin12, O Sheils1 and JJ O’Leary1.

1. Histopathology, Trinity College, Dublin 2, Ireland .
2. Pathology Dept, Coombe Women and Infants University Hospital, Dublin 8, Ireland.

Prostate cancer is the second leading cause of cancer deaths in men. Stem-like cells have been identified in several malignancies including prostate cancer and are thought to drive primary tumorigenesis through self-renewal and differentiation. Additionally, persistence of stem cells post-therapeutic intervention has been proposed as an explanation for metastasis and recurrence. Holoclones are a tightly packed clone of small cells generally thought to contain stem cells and progenitors. miRNAs are recently discovered small family of regulatory molecules that are associated with various malignancies. The aim of this study was to identify a profile of prostate cancer-associated miRNAs, and to derive holoclones from prostate cancer cell lines and to characterise the miRNA profile of these stem-like cells. In this study, meta-analysis was carried out to compare existing data on miRNA expression in prostate cancer and data was analysed using miRGen and miRNApath. The expression of a panel of known human miRNAs, was assessed in a group of prostate cell lines PWR-1E (normal), LNCaP (metastatic carcinoma), PC-3 (non-metastatic adenocarcinoma) using a quantitative Real Time TaqMan PCR method. Meta-analysis identified common miRNA species in prostate cancer and the predicted gene targets and pathways were also assessed. Putative holoclones were generated from cell lines (LNCaP, PC-3) using a high salt-soft agar assay and LNCaP putative holoclones were maintained for 24 days and PC-3 holoclones were maintained for 6 days (LNCaP cell line represents a metastatic prostatic carcinoma and should contain a higher number of cancer “stem” cells). miRNA profiling was performed using 380 individual assays based on Stem Looped Primer PCR reactions. Analysis of the data showed unique miRNA populations varied between each of the cells profiled. Future work will consist of further analysis on the expression of human miRNAs in prostate cancer cell lines and holoclone derived stem cells in order to identify prostate cancer “stem” cell-specific miRNA populations.

Acknowlegements: Prostate Cancer Research Consortium.

 

Silencing of HPV Viral Oncogenes E6 and E7 in Cervical Cancer.

1,2 Spillane CD, 1,2Kehoe L, 1Sheils O, 1,2Martin CM, 1,2O’Leary JJ.
1 Dept. of Histopathology, Trinity College Dublin, Ireland.
2 Dept. of Pathology, The Coombe Women & Infants University Hospital, Dublin, Ireland.

HPV is the main etiological agent in cervical cancer (CC), with high-risk HPV type 16 and 18 detected in more than 70% of CC. Integration of high-risk HPV genomes and expression of the viral oncogenes, E6 and E7, are critical steps in the development of CC. The aim of this project was to use short interfering RNA (siRNA) targeted towards the E6/ E7 oncogenes. HPV16 transformed SiHa cells are being used as a model system. The most efficient transfection agent for the cells was established using a high throughput 96-well set up. With controls a central issue in RNAi experiments it was essential to initially identify a negative control siRNA for the system and optimise transfection conditions for a positive control siRNA. A panel of negative controls were obtained. Using the optimal transfection agent, Lipofectamine RNAiMax, the cells were transfected with these siRNA and by means of E7 and E6 specific TaqMan® PCR their ability to affect the HPV16 genome was assessed. Subsequently, the optimal knockdown conditions for the positive control siRNA, GAPDH, were determined. Silencing of on average 87% at the RNA level has been achieved using the optimal conditions. The cells were next transfected with siRNA specific to HPV16 E7. To determine whether the expression of E7 and E6 RNA was altered, E7 and E6 specific Taqman® PCR was performed. The effect at the protein
level was examined by investigating the expression level of E6 and E7 targeted proteins, p53, p21 and Rb. In excess of a 70% reduction in RNA levels of E7 has been achieved, with a concomitant silencing of HPV16 E6. We describe an RNAi approach which leads to the suppression of HPV16 E6 and E7. This approach may have a potential role for genespecific therapy in HPV16 associated CC.

 

Characterisation of Early Stemness Regulation in Teratocarcinoma Stem Cells

S Elbaruni, M Gallagher, CCBB Heffron, S Guenther, R Henfrey, C Martin, O Sheils & JJ O’Leary
Department of Pathology, Coombe Women and Infants University Hospital, Dublin 8, Ireland.
Department of Histopathology, Trinity College, Dublin 2, Ireland

Background: It is widely accepted that extensive self-renewal and differentiation (defined as ‘stemness’) of cancer stem cells (CSCs), progenitor cells required for normal tissue renewal that appear most likely cells of origin of tumours, may drive tumourigenesis. Furthermore, persistence of CSCs post-intervention may explain metastases and recurrence. Identification of CSCs in brain, breast, prostate, head and neck and ovarian tumours imply that CSCs are key players in malignancy. However, clinical inhibition of CSC stemness has not been achieved to date. As CSCs clearly mirror many aspects of normal stem cells (NSCs) of comparable potency and are functionally pluripotent (can self-renew and differentiate to produce mature cell types), we hypothesised that CSCs were characterised by aberrant regulation of differentiation rather than of differentiation itself. Addressing this, we have identified novel CSC regulatory events through whole-genome analysis of self-renewal and early differentiation in CSCs.

Design: Human teratocarcinoma (‘classical stem cell’ gonadal tumours) CSCs originally derived from well (pluripotent) and poorly-differentiated (nullipotent) tumours were stimulated to differentiate for 3 days via addition of retinoic acid. Whole-genome array analysis was subsequently performed on undifferentiated and differentiated samples and profiles validated through further analysis of 50 genes of interest by TaqMan real-time PCR analysis. Validated gene expression profiles were bioinformatically compared to published hES data, permitting identification of gene events
exclusive to CSCs.

Results: As stemness analysis has generally been conducted at approximately 1 week differentiation, we assayed CSCs at 3 days differentiation to identify early CSC stemness regulatory genes. As hypothesised, resultant data was enriched with novel regulatory genes previously unassociated with CSC stemness including regulators of stemness pathways such as Wnt, Snail, Notch and Shh and a novel marker of mesodermal differentiation in CSCs, ENO3. We hypothesise that these early stemness genes regulate key downstream stemness genes and pathways and postulate that specific CSC-targeting, in a manner not affecting NSCs, can now be achieved by their functional knockdown.

Conclusion: We have identified gene expression profiles enriched for genes involved in early cancer stemness. We believe that this new understanding of CSC biology will facilitate achievement of targeted removal of CSC-stemness in a manner applicable to cancer therapeutics.

 

Elucidation of the Role of the P16INK4A Pathway in the Cell Cycle and its Relationship with the Human Papilloma Virus in Cervical Cancer.

Kehoe L, Spillane CD, Gallagher M, Martin C, O’Leary JJ.
Department of Histopathology, Trinity College Dublin, Ireland.
Department of Pathology, Coombe Women and Infant’s University Hospital, Dublin, Ireland.

Unravelling effects of high risk HPV oncoproteins, E6 and E7, in cervical cancer (CC) has led to the discovery of proteins that are dysregulated and therefore potential biomarkers. One of these is p16INK4A (p16). Although p16 protein expression is currently being used diagnostically as a marker of cervical dysplasia, very little is known about its involvement in the cell cycle. We aim to determine the cause and effects of p16 up regulation in CC, using the HaCaT cell line, which has previously been reported as p16 null due to promoter hypermethylation. We have recently discovered HaCaT cells are expressing p16 mRNA using Taqman® PCR and that the p16 promoter is unmethylated via Methylation specific PCR. However western blots have indicated a lack of detectable protein expression. We hypothesize that p16 mRNA is degraded before it reaches the polysome. Preliminary examination of mRNA from polysome extracts has been performed. Results indicate the presence of a small amount of p16 message at the polysome. This is possibly due to interference by small RNAs such as natural anti-sense transcripts (NAT) and or microRNAs (miRNA). Future studies will involve examination of expression of the products of the INK4A/ARF locus and polysomal extracts from other CC cell lines. Taqman® PCR will be used to examine NAT expression and miRNA profiling of the HaCaT cell line versus other CC cells and normal cervix will be performed. This will determine if NATs or miRNAs are a regulatory system for control of the INK4A/ARF locus and also if HPV effects this mechanism.

 

Evaluation of the Diagnostic Potential of Molecular Biomarkers of Cervical Cancer.

Lynda McEvoy, Katharine Astbury1,2, Orla Sheils2, Cara M Martin1,2, John J O’Leary1,2.

1. Coombe Women’s Hospital, Dolphin’s Barn, Dublin 8, Ireland.
2. Department of Histopathology and Morbid Anatomy, Trinity College Dublin, Dublin 2, Ireland.

Cervical cancer is the second most common malignancy among women in the world. The diagnosis of Cervical Cancer is based on cellular morphology, however this is a subjective process, associated with a high level of false positive and false negative results. Molecular biomarkers are molecules which are differentially expressed in cancer compared to normal tissue, and may be used as an adjunct to cellular morphology in order to differentiate between normal and cancerous or precancerous tissue. The aim of this study was to examine the mRNA and protein expression levels in Cervical Cancer and Precancer of three potential biomarkers of Cervical Cancer: Topoisomerase IIa, Survivin and Nuf2. Immunohistochemistry for TopoIIa, Survivin and Nuf2 was carried out on a range of Normal, CIN1, CIN2, CIN3 and cGIN cases. Quantitative Real Time-PCR analysis of mRNA expression was carried out for TopoIIa and Survivin on a subset of these cases. An increase in TopoIIa protein and mRNA expression was observed in CIN1, CIN2, CIN3, SCC and cGIN cases compared to normal cervical epithelium, and an increase in mRNA expression was observed with increasing grade of squamous abnormality. Survivin protein expression of varying intensity was observed in all cases while Survivin mRNA expression was increased in CIN, SCC and cGIN compared to normal cervical epithelium, and increased with increasing grade of squamous abnormality. Nuf2 protein expression was absent from normal cervical epithelium and SCC, however was present with varying intensities in all cases of CIN1, CIN2 and some cases of CIN3. The results of this study indicate that TopoIIa may be used as an adjunct to cellular morphology in order to differentiate between low grade and high grade CIN and that Nuf2 may be a novel marker of precancerous lesions.

 

Gene expression profiling of peripheral blood during in vitro fertilisation treatment reveals
predictive markers of pregnancy.

Cathy Allen, Cara Martin, Edgar Mocanu, Michael Geary, John J O’Leary.

Background: Reproduction is a fundamental biological process central to the survival of humans, yet the actual biological and molecular pathways involved remain poorly understood. Infertility affects more than 80 million couples worldwide, and it is estimated that one in seven UK couples require medical help for fertility problems. With average clinical pregnancy rates following in vitro fertilisation (IVF) for UK patients remaining under 30% per treatment cycle, there is a clear need to identify early predictors of IVF treatment success and implantation failure. In this study, we examine the functional transcriptome pattern in the early stages of IVF induced pregnancy and non-pregnant controls, to define sets of predictive biomarkers which identify implantation events, maintenance of pregnancy and successful / non-successful pregnancy outcome.

Study Design: In this study, we examine the global gene expression patterns in RNA extracted from peripheral
blood samples taken at 8 different time points during the peri-conceptual period and early stages of IVF cycle, from 5 women who achieved clinical pregnancies, 3 women who had implantation failure, and 3 subfertile women who conceived spontaneously.

Total RNA was extracted from 3ml of whole blood collected in TempusTM RNA collection Tubes, and extracted
using the ABI PRISM™ 6100 Nucleic Acid PrepStation. Gene expression analysis was performed (according to the manufacturer’s instructions) using AB high density microarray system the 1700, and the AB Human Genome Survey Microarrays, which simultaneously screens a single RNA sample for expression of over 30,000 human genes. Data analysis was performed using R statistical package, molecular function and biological process analysis was performed using Panther.

Results: Using this global gene expression profiling approach, we found that 128 genes showed a differential
expression of more than 2-fold in early clinical pregnancy (7 weeks gestation) compared with a non-pregnant state (pituitary desensitization) (p < 0.05). 103/128 genes were up-regulated and 25/128 were down-regulated. The most over-represented molecular pathways include angiogenesis, endothelin signaling, inflammation, oxidative stress, VEGF, and pyruvate metabolism.

A panel of 200 genes were differentially expressed at a pre-treatment time-point in a group of women who became pregnant following IVF compared to those who did not become pregnant (p<0.05). 134/200 genes were up-regulated and 76/200 genes were down-regulated. The most significant molecular pathways represented by this cohort of differentially regulated genes were; angiogenesis, blood coagulation, endogenous cannabiniod, inflammation, and Wnt signaling pathway. In addition transcripts associated with cysteine biosynthesis were differentially expressed in the early post-conceptual period in women with successful implantation compared with women with implantation failure.

When gene expression profiles for first trimester IVF conceptions were compared with spontaneous conceptions in subfertile women, 237 genes showed a differential expression of more than 2-fold (p<0.05). 67/237 genes were upregulated and 170/237 were down-regulated. The most significant molecular pathways represented by these genes involved Heterotrimeric G protein signaling.

Conclusions: This is the firs time that gene expression profiles of the peripheral blood during in vitro fertilization are reported. The expression profiles represent a transcriptome signature for early human pregnancy that is influenced by the trimera of mother, fetus, and placenta. Gene expression profiles prior to IVF treatment are predictive of treatment success or failure, and offer valuable potential in clinical decision-making for future IVF patients. The significant degree of gene expression disturbance between assisted and natural conceptions is of concern and highlights the need for further efforts to optimize IVF protocols.

 

Potential Biomarkers of Cervical Cancer Identified by Gene Expression Profiling and TaqMan Polymerase Chain Reaction

Katharine Astbury 1,2, Cara M Martin 1,2, Lynda McEvoy, Orla Sheils 2, Sean Daly 1, John J O’Leary 1,2.

1. Coombe Women’s Hospital, Dolphin’s Barn, Dublin 8, Ireland.
2. Department of Histopathology and Morbid Anatomy, Trinity College Dublin, Dublin 2, Ireland.

Cervical cancer is the second most common female malignancy worldwide. While screening programmes using the Papanicolaou smear have reduced the incidence of squamous cell carcinoma in the developed world, the occurrence of both false positive and false negative screening results and the rise in incidence of cervical adenocarcinoma has led to the quest for potential biomarkers to enhance diagnostic accuracy and facilitate early diagnosis of the disease. In addition, advances in the area of pharmacogenomics have led to the identification of potential gene targets for therapy in several cancers. Gene expression profiling experiments offer the opportunity to interrogate thousands of genes simultaneously and thus potentially significantly enhance the rate of discovery of potential novel biomarkers, when compared to traditional approaches such as immunohistochemistry. In this study, we used gene expression profiling to identify genes that are differentially expressed in cervical cancer cell lines compared to normal cervical tissue. TaqMan PCR was then used to validate a number of the target genes identified. In addition, to a number of previously evaluated biomarkers, we have identified a number of genes, including several involved in cell cycle regulation, which are over-expressed in cervical cancer. The next step will be to evaluate the clinical utility of these
genes as biomarkers of cervical cancer.

 

Comparison of HPV detection technologies; Hybrid capture 2, PreTect™ HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens.

Helen Keegan a, *, Jamie McInerneya, b, Loretto Pilkingtona, Petter Grønnc, Ivan Silvac, Frank Karlsenc, Noel Bolgera, Catriona Logana, d, Liv Furuberge, John O’Learya, b, Cara Martina, b

a Department of Pathology, Coombe Women and Infants University Hospital, Dublin 8, Ireland.
b Department of Histopathology, University of Dublin, Trinity College, Dublin 2, Ireland.
c Norchip AS, 3490 Klokkartsua, Norway.
d Microbiology Department, Our Lady’s Childrens Hospital, Crumlin, Dublin 12, Ireland. e SINTEF ICT, MiNaLab Facility, Gaustadalleen 23C, 0373 Oslo, Norway.

Human papillomavirus (HPV) testing using molecular methods in liquid based cytology specimens (LBC) may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6/E7 mRNA method PreTect™ HPV-Proofer in cytological specimens (n=299).

LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205/299) of the cases were positive by hc2 and 38% (112/299) of the cases were positive by PreTect™ HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories; (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect™ HPV-Proofer.

The sensitivity and specificity of PreTect™ HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively.

The relatively low detection rate observed by PreTect™ HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect™ HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.

 

Development of a Point of care Diagnostic chip for detection of active oncogenic HPV infection in cervical smear specimens. The MicroActive Project.

C. Martin1, H. Keegan1, J. L. Solli2, A. Gulliksen2, T. Baier3, R. Gransee3, T. Hansen-Hagge3, M. Mielnik4, I-R. Johansen4, L. Furuberg4, L. Rigger5, P. Koltay6, F. Karlsen2 and O’Leary1

1. Coombe Women & Infants University Hospital, Dublin, Ireland.
2. NorChip AS, Klokkarstua, Norway.
3. Institut für Mikrotechnik Mainz GmbH, Germany.
4. SINTEF ICT, Oslo, Norway.
5. Department of Microsystems Engineering (IMTEK), Albert-Ludwigs-Universität Freiburg, Germany, 6BioFluidix GmbH, Freiburg, Germany.

Cervical cancer is the second most predominant form of cancer among women worldwide. The primary risk factor for developing cervical cancer is infection with Human Papillomavirus (HPV), with HPV types 16 and 18 detected in greater than 70% of cervical carcinomas. Detection of persistent HPV infection with oncogene (E6E7 mRNA) expression has the potential to identify at an early stage women who will require treatment for the disease. Against this background, within the MicroActive Project we have developed a state of the art “point of care” diagnostic device which is an automated integrated chip-based platform which has the capability to extracts nucleic acids from cervical smear specimens and simultaneous detects multiple types of active oncogenic HPV infection. This combined system will ultimately serve as a point of care system for the detection of gene expression directly in a physician’s office, avoiding the often delayed analysis by a specialised laboratory. This is the first system of this kind to be developed and tested on clinical specimens. The technology used is state of the art and will pave the way for future diagnostic tests.

Furthermore the developed system has capabilities far beyond cervical cancer and with small modifications can be adapted to other applications where it is desirable to analyse complex biological samples “in the field” and on a short timescale. This includes for example Foodstuff analysis / animal feed control, personalised medicine and forensic analysis.

This work was funded by the European Commission within its 6th Framework Programme under contract no. IST-NMTCT- 2005-017319.

 

An Integrative Transcriptome Map of Recurrence in Ovarian Cancer.

A Laios, SA O’Toole, BL Sheppard, R Flavin, N Gleeson, T D’Arcy, EPJ McGuinness, M Ring, C Martin, O Sheils
and JJ O’Leary.

Departments of Obstetrics and Gynaecology and Histopathology, Trinity College, St. James’s Hospital and Coombe Women’s Hospital, Dublin.

Background: Ovarian cancer is a devastating disease partly because conventional chemotherapy ignores aspects of tumour biology. Recurrence represents the “true killer” among ovarian cancer patients and identification of novel molecular markers present in recurrent tumours is urgently required.

Methods: We carried out gene expression profiling between primary and recurrent ovarian cancers and identified potential biomarkers of recurrence using Applied Biosystems array 1700.

Results: We identified distinct patterns of gene expression between primary and recurrent ovarian cancers from different patients with the same histology and from the same patients with different histology. Selected targets were validated and correlated with microarray results. Signatures from both cohorts were also validated against an independent set of serous papillary ovarian adenocarcinomas. Notably, upregulated genes in the recurrent compared to primary tumours in both cohorts, segregated in the same gene families. Included among these genes are S100B and S100A8 belonging to the S100 family of calcium binding cytoplasmic proteins, TJP3 and CLDN16 belonging to the family of tight junction proteins, BTC and NRG2 belonging to the family of EGFR ligands, and interleukin receptors IL1R2 and IL27RA.

Conclusions: Collectively, our data proposes an integrative model for recurrence in ovarian cancer, in which tumour cells during relapse produce adhesion molecules to mediate attachment, cytokines and inflammatory mediators to stimulate survival and a variety of growth factors bound to their cognate receptors to fully proliferate in order to confront and modulate their immediate environment. Some of the mechanisms involved in recurrence could be specific to the drugs used.

 

Altered Expression of miR-223 and miR-9 in Recurrent Ovarian Cancers may Target the FGF Family

Laios A, O’Toole SA, Flavin R, Kelly L, Martin C, Ring M, Gleeson NC, D’Arcy T, McGuinness EPJ, Sheils O, Sheppard BL, O’Leary JJ.

Departments of Obstetrics and Gynaecology and Histopathology, Trinity College Dublin, St. James’s Hospital and Coombe Women’s Hospital, Dublin.

Background: Our transcriptome profiling has previously identified a gene signature which may be representative of recurrent/chemoresistant ovarian cancer. Our recent profiling of 180 miRNAs in the same cohort of recurrent versus primary serous papillary adenocarcinomas identified miR-223 and miR-9 as the top up and downregulated miRNAs respectively.

Methods: Independent validation of miR-223 and miR-9 was carried out by extracting total RNA from 12 primary and 8 recurrent fresh frozen ovarian tumours using the Ambion mirVana™ miRNA isolation kit. miRNA expression levels were examined using the Applied Biosystems stemloop RT/PCR kit (ABI). Quantification of recurrent samples was carried out relative to primary using the DDCt method. Let-7a was used as an endogenous control.

Results: miR-223 was upregulated in recurrent versus primary fresh frozen tumours and miR-9 downregulated by more than two fold change.

Conclusions: miR-223 and mir-9 are predicted to target members of the Fibroblast Growth Factor family which was identified as a target in our transcriptome study. This gene family may be associated with the acquired metastatic capacity or recurrence pattern of advanced epithelial ovarian cancers and warrants further investigation.

 

Evaluation of MMP-9 inhibitor and cisplatin cytotoxicity in a chemoresistant ovarian cancer cell line using high content screening (hcs) cell-based assays.

A Laios, SA O’Toole, B Png, R Flavin, L Kelly, T D’Arcy, E McGuinness, N Gleeson, B Sheppard, O Sheils, JJ O’Leary. Department of Obstetrics and Gynaecology/Histopathology, Trinity College Dublin, St James’s Hospital and Coombe Women’s Hospital, Dublin, Ireland.

Background: Resistance of recurrent disease to cytotoxic drugs limits long-term treatment success against
ovarian cancer. In a previous transcriptome approach, we identified MMP-9 as a potential marker of recurrence/
chemoresistance. We hypothesized that using a chemical inhibitor of MMP-9 (MMP-9i) alone or in conjunction with cisplatin the chemoresistance phenotype seen in ovarian cancer cells could be overcome.

Methods: A2780cis (cisplatin-resistant) human ovarian carcinoma cell line was used. The cytotoxic effect of MMP- 9i and MMP-9i+cisplatin in combination was determined using the HCS multiparameter cytotoxicity 1 Hit Kit by Cellomics. Cells were plated in triplicate in 96-well plates overnight and then treated with various concentrations of drugs for 3, 6 and 24 hours. They were then stained with a three-in-one fluorescent dye to image nucleus, cell permeability and lysosomal pH respectively. Image acquisition analysis was performed by using the Cellomics KineticScan (an automated fluorescent microscope) and the optimized software. The average intensity was used to quantify changes.

Results: MMP-9i alone had a cytotoxic effect in cisplatin-resistant at high doses (260-520μM) at 3h, 6h and 12h
time points but also at an early time course (3h) even at sublethal dose (56µm). The combined treatment of MMP- 9i+cisplatin enhances cytotoxicity compared to cisplatin alone. Preincubating cells with MMP-9i at all doses were significantly cytotoxic at 3h but not 6h prior to treating with cisplatin.

Conclusion: Specific MMP-9i might have a place in treatment of recurrent/chemoresistant ovarian cancer that their role in-vivo remains to be tested.

 

The evaluation of the Nicotine Metabolite assay on the Immulite 2000 immmunoassay analyser and its use in urine in pregnancy.

O’Kelly RA., Lynch CM., Regan CL. and O’Leary JJ.

Aims: Nicotine is metabolised in the liver to cotinine, trans-3’-hydroxycotinine (the major renal excretion product of nicotine) and other metabolites and these are specific for the detection of smoking. It is intended to evaluate The Immulite 2000 Nicotinine Metabolite assay and its application in urine.

Method: The assay is a solid-phase competitive chemiluminescent immunoassay, linear between 10 and 500 ng/mL with an auto-dilution facility and uses a highly specific polyclonal rabbit anti-cotinine antibody.
Assay performance for precision, functional sensitivity, linearity on dilution was evaluated using control material and urine. Urine samples from 120 pregnant women in the third trimester were analysed for Nicotine Metabolite.

Results: Intra-assay precision at 36 ng/mL and 8506 ng/mL resulted in CV of 5.2% and 5,5% respectively (n=10). Inter-assay precision over 20 runs resulted in CVs of 10.7% at 17.5 ng/mL, 9.9% at 53 ng/mL and 9.8% at 8975 ng/ mL. Functional sensitivity investigation showed a CV of 9.4% (n = 5) for a sample containing 11.7 ng/mL Nicotine Metabolite (the stated detection limit is 10 ng/mL). Linearity on dilution using the automatic dilution facility resulted in recoveries of 96-99% for the 1/100 dilution compared to the 1/40 dilution.

Urine samples from 120 pregnant women were analysed: 60 women had declared themselves as smokers and 60 as non-smokers. 32 non-smoking women had a Nicotine Metabolite of <10 ng/mL, while 28 had a value of 10-60 ng/ mL. 15 of this latter group were known to have been exposed to passive smoking and their mean value was 31 ng/mL (range 12 - 60). The average Nicotine Metabolite value in the smokers was 5565 ng/mL with a range of 25–18891 ng/ mL.

Conclusion: The assay performs within the manufacturer’s specifications. The values obtained in urine samples appear to reflect smoking status. Passive smoking results in small but measurable quantities of Nicotine Metabolite in urine.

 

Automation in Cervical Cytology

N. Bolger1, R Cullen2, M Griffin2, JJ O’Leary1

1. Department of Cytopathology, Coombe Women’s Hospital, Dublin.
2. Department of Cytopathology, St. James Hospital Dublin, Ireland.

Cervical cancer is the second most common female malignancy worldwide. In Ireland, in 2001 there were 185 new cases of cervical cancer diagnosed and almost 70 deaths. A well-defined pre-malignant phase (cervical intraepithelial Neoplasia/CIN) exists and this can be detected by screening using cervical cytology. The introduction of cervical screening programmes in other countries has dramatically decreased the incidence of cervical cancer. However, to date no comprehensive national screening programme has been introduced in Ireland. Despite this, opportunistic screening in Ireland has detected significant numbers of cases of CIN in Irish women. In 1986, 811 cases of high-grade CIN were diagnosed and this number rose to 1178 in 2001, indicating that this is a common and increasingly prevalent problem.

As up to 30% of high-grade CIN lesions progress to invasive cancer over 10 years, this rising incidence of CIN would suggest that we can anticipate a significant increase in the numbers of cervical cancers being diagnosed in the next decade unless a comprehensive screening programme is introduced.

The Irish Cervical Screening Research Consortium (ICSRC), with significant hospital, academic, biotechnology, health economic, epidemiological psychological and national agency support was set up in 2006 to improve the quality of a cervical screening programme in Ireland and seeks to introduce major innovations in the cervical screening process through the introduction of Automation, ancillary viral testing for use in management triage and the development of objective biomarkers and biosensors for use as prognostic indicators of disease outcome.

Project 1 of 8 overall projects implemented by the ICSRC was designed to look at Automated screening in routine cytology practice, utilising the Imager technology from Cytyc Corporation. The project uses two approaches: A. a randomised controlled trial (RCT) of automated versus manual screening and B. a within-subject trial arm.

Today’s talk will describe the experience of the Coombe Women and Infants University Hospital (CWIUH) with the Imager technology since its introduction in 2004 and also present current data related to Project 1.

 

Sex Steroids used in Hormone Treatment alter Coagulation Gene Expression in Cultured HUVEC.

J.F. Brosnan, B.L. Sheppard, J.J. O’Leary, L.A. Norris.
Obstetrics and Gynaecology, University of Dublin, Trinity College, Dublin, Ireland. Department of Histopathology, University of Dublin, Trinity College.

Introduction: Hormone therapy modulates plasma levels of proteins that regulate blood coagulation, however, the underlying mechanisms responsible are unclear. The aim of the study was to investigate the in-vitro effects of 17b estradiol, its metabolites and norethisterone acetate (NETA) on coagulation and fibrinolytic gene expression in cultured Human Umbilical Vein Endothelial Cells
(HUVECs).

Methods: Cultured HUVECs were treated with either 17 b estradiol(10nM), Estrone (10nM), 2-hydroxyestradiol(10nM), NETA (10−8M) and NETA (10−8M)/17b-estradiol (10nM) or vehicle control for 24 hours. Real time Taqman PCR was used to examine the mRNA expression levels of Thrombomodulin (TM), Endothelial Protein C Receptor (EPCR), Tissue Factor (TF), Tissue Factor Pathway Inhibiton (TFPI), tissue Plasminogen Activator (tPA) and plasminogen inhibitor (PAI-1). Protein expression in the culture media was measured using Enzyme Linked Immunosorbent Assay(ELISA) techniques.

Results: TF mRNA expression was significantly increased in HUVECs treated by NETA alone compared to vehicle control (p<0.01) or 17b estradiol (p<0.004). TFPI mRNA expression was significantly increased in cells treated with NETA alone (p<0.012) and cells treated with both 17b estradiol and NETA (p<0.039). Expression of mRNA tPA was also increased following NETA alone and NETA/17b estradiol treatment, however the increase did not reach statistical significance (p<0.055). Treatment with 17b estradiol, estrone and 2-hydroxyestradiol showed small non significant increases in expression of TFPI, tPA and TF. Thrombomodulin, EPCR and PAI-1 mRNA expression in HUVECs was not significantly affected by any of the treatments studied. Similarly, protein expression in the media of treated cells was not significantly altered.

Conclusions: These results show that NETA can significantly influence the TF/TFPI gene expression. These effects may contribute to the increased coagulation activation reported in postmenopausal women using hormone therapy.