Abstracts presented to the Blair Bell Research Society, 12th–13th October 2006, Leicester

Local delivery of adenovirus VEGF to the uterine arteries increases vessel relaxation and uterine artery blood flow in the sheep

AL David,1 B Torondel,2 I Zachary,2 MJ Ramirez,2 SM Buckley,3 T Cook,4 M Boyd,5 CH Rodeck,1 J Martin,2 D Peebles1

1 Obstetrics & Gynaecology and 2 Centre for Cardiovascular Biology & Medicine, Royal Free and University College London Medical School, London; 3 Gene Therapy Research Group and 4 Pathology, Imperial College School of Medicine, London; 5 BSU, Royal Veterinary College, London

Impaired materno-placental perfusion can lead to pre-eclampsia and fetal growth restriction. We investigated whether local expression of VEGF in the uterine artery would increase uterine artery blood flow (UABF) and alter vascular tone.

We injected adenovirus vectors containing the VEGF or β-galactosidase gene (Ad.VEGF-A or Ad.lacZ, 5 × 1011 particles) into the left and right uterine artery of five ewes at laparotomy (97 days gestation, term = 145 days); the investigators were blind to the side of VEGF injection. Mean UABF, measured by Doppler sonography 5 days after injection, significantly increased after VEGF-A injection when compared with before injection (6.81±4.56 to 22.14±12.37 ml/sec, p=0.005); Ad.lacZ had no significant effect. Compared with Ad.lacZ vessels, Ad.VEGF-A transduced vessels were significantly less responsive to phenylephrine (Emax 148±10.9 vs 228.2±27.5, p<0.05) and had significantly increased relaxation to bradykinin (pD2 values 9.11±0.01 vs 8.65±0.11, p<0.05). Adenovirus mediated local VEGF overexpression increases UABF and uterine artery relaxation.

Elevated first-trimester maternal serum Ischemia Modified Albumin – evidence for hypoxia-driven placental growth, and a marker for the development of severe pre-eclampsia

AT Papageorghiou, F Prefumo, DC Gaze, R Sharma, SJD Brecker, PO Collinson, B Thilaganathan

St George’s Hospital, Tooting, London

Hypoxia is believed to play a pivotal role in physiological trophoblast development and pathogenesis of pre-eclampsia. Ischemia modified albumin (IMA), a novel protein used in the diagnosis of cardiac ischemia, was measured in healthy women with singleton pregnancies at 11–14 weeks. Maternal serum IMA concentrations in those subsequently developing severe pre-term pre-eclampsia (n=19) were compared to controls with normal outcome at term (n=45). IMA levels in pregnancy were higher than non-pregnant normal ranges; were significantly higher in women with subsequent pre-eclampsia when compared to those with normal pregnancy outcome; and were significantly correlated with first-trimester uterine artery RI. Supra-physiological levels of IMA in early normal pregnancy support the hypothesis that trophoblast development is stimulated by a hypoxic intrauterine environment. High first trimester IMA levels and uterine artery resistance indices in severe pre-eclampsia indicate that trophoblast does not respond normally to increased placental hypoxia. This may be a consequence of reperfusion oxidative damage.

Changes in peripheral arterial pulse wave parameters in normal human pregnancy

A Khalil, D Cooper, K Harrington

Homerton University Hospital, Homerton Row, Hackney, London E9 6SR

Outside pregnancy, analysis of the peripheral arterial pulse wave provides valuable information in hypertension and vascular disease. It may also distinguish the vascular changes of established pre-eclampsia (PET) from normal. However, data on the normal changes in pregnancy are sparse and need to be accurately defined before the possibility of screening for PET can be explored. The radial artery pulse waveform was analysed in 168 healthy pregnant and 20 non-pregnant women using applanation tonometry. Augmentation pressure (AP) and Augmentation Index (AI) were calculated. Mean AP (3.32 versus 6.8) and mean AI (15.8 versus 24.6) were significantly lower in pregnancy than non-pregnancy (p<0.001). There was no significant difference in AP or AI across the three trimesters. Our work has defined the normal changes in peripheral arterial wave characteristics in pregnancy, confirming a fall in vascular resistance in pregnancy. These data provide the foundation for further investigation into the potential role of the technique in diagnosis and prediction of vascular disease in pregnancy, in particular PET.

Correlation between MHC class II expression on cord blood monocytes and outcome in term and preterm neonates

J Lloyd, M Aziza, M Allen, N Klein, D Peebles

Centre for Perinatal Brain Protection and Repair, UCL, London, United Kingdom, WC1E 6HX

MHC Class II expression on monocytes is necessary for immune competence and can be down-regulated following inflammatory stimuli. To investigate the relation between pro/antinflammatory balance and neonatal outcome we measured monocyte MHC Class II expression, using flow cytometry, in cord blood from 106 fetuses born at term after labour or elective delivery, after preterm labour (PTL) or prolonged pre-labour rupture of membranes (PPROM) at <32 weeks. MHC Class II expression was not affected by term labour, but was lower in neonates born after PTL or PPROM than term infants, p<0.001. It was negatively associated with death in premature neonates, median 44.74% vs 72.0% in survivors, p<0.05, and was reduced in term infants with sepsis, p<0.0001. Low levels of monocyte MHC Class II expression, perhaps as a result of exposure to an inflammatory stimulus in utero, may partly explain the increased susceptibility to infection observed in premature infants.

Angiotensin II Type 2 receptor (AT2) RNA expression in human placentae

CL Tower, C Popescu, HA Lacey, M Desforges, PN Baker, JD Aplin

Maternal and Fetal Health Research Centre, Division of Human Development, University of Manchester, St Mary’s Hospital, Manchester M13 0JH

The AT2 receptor is implicated in pre-eclampsia but data on its presence in human placenta are conflicting. Tissue from term deliveries and social terminations (8-18/40) was obtained with patient consent and ethic committee approval and RNA extracted. Real time RT-PCR was conducted on the Stratagene MX4000 using SYBR green detection. Alternative AT2 transcripts have been described, necessitating location of primers within a single exon. Direct PCR conducted on RNA was used to exclude samples (n=10) contaminated with genomic DNA. Positive and negative controls were included. 15 of 24 placental samples contained AT2 receptor mRNA. Of these, 5 were collected from term and 10 were from terminations. Of the 9 negative samples, 6 were from terminations (all<12/40) and 3 from term deliveries. This study suggests the expression of AT2 receptor mRNA in human placenta in all three trimesters. Ongoing work is investigating placental AT2 receptor protein using immunohistochemistry and Western Blotting.

The effect of oxygen on the generation of active soluble factors from placental villous explants

NJ Robinson, NA Sheikh, JD Aplin, PN Baker, IP Crocker

Division of Human Development, University of Manchester, Manchester M13 0JH, UK

In pre-eclampsia a hypoperfused placenta secretes a circulating factor(s) into the maternal circulation, triggering endothelial dysfunction. We have optimised serum-free conditions for the culture of placental villous explants and the subsequent testing of liberated factors on primary endothelial cell cultures. Explants from normal pregnancies were exposed to 20%, 6%, and 1%O2 to mimic physiological and non-physiological intervillous oxygen tensions. Media collected from viable explants between 3 and 4 days in vitro were applied to human uterine microvascular endothelial cells. Medium conditioned by hypoxic explants caused a significant decrease in endothelial cell ATP and mitochondrial dehydrogenase activity (MTT test) suggestive of a reduced endothelial cell metabolic rate. There was no change in the rates of cell death as measured by LDH release and ADP:ATP ratio. Thus hypoxia (1%O2) stimulates the release of factors from the placenta which influence the metabolic status of microvascular endothelial cells in vitro. These potentially pathogenic factors are now being characterised.

HDAC inhibition, human uterine contractility and preterm labour

MP Hehir,1 AM Sharkey,1 SC Robson,2 GN Europe-Finner,2 JJ Morrison1

1 Department of Obstetrics and Gynaecology, University College Hospital, National University of Ireland Galway, Galway, Ireland; 2 School of Surgical and Reproductive Sciences, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne

There is now accruing data to indicate that HDAC inhibitors such as Trichostatin A (TSA) and Valproic Acid (VPA) may potentiate the maintenance of uterine quiescence during pregnancy via increasing the expression of myometrial hCG receptors and at the same time decreasing the expression of COX-2 in human amnion cells thus decreasing uterine prostaglandin production. At present there are no functional data to indicate that these agents can affect myometrial activity in vivo. The aim of this study was to evaluate the effect of TSA and VPA on human uterine contractions, and hence their potential role as tocolytic agents.

Biopsies of human myometrium were obtained at elective caesarean section (n=18). Dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of TSA in the concentration range of 1 nmol/L to 100 mmol/L and VPA (100nmol/L-1mmol/L). Two sets of control experiments were performed simultaneously as follows: 1. Strips exposed to either physiological salt solution (PSS) only (for spontaneous contractions) or 0.5 nmol/L oxytocin; 2. Strips exposed to PSS/oxytocin and vehicle for TSA or VPA. TSA and VPA exerted a potent and cumulative inhibitory effect on spontaneous (n=6) and oxytocin-induced contractions (n=6), compared to control strips. No significant differences were found between control 1 and control 2 for both spontaneous and oxytocin induced contractions.

The HDAC inhibitors TSA and VPA exerted a potent inhibitory effect on human uterine contractions. This raises the possibility that this new class of compounds may have tocolytic potential, in addition to their current clinical indications. Future work will focus on the genomic and non-genomic mechanisms of the effects observed.

A randomised controlled trial of feeding in labour

B Liu, G O’Sullivan, D Hart, M Waterstone, AH Shennan

Maternal & Fetal Research Unit, King’s College London; Department of Anaesthesia, St Thomas’ Hospital, London; Department of Obstetric and Gynaecology, Queen Mary’s Hospital, Sidcup

Although a number of studies have been conducted to investigate the influence of oral intake on labouring women, limited sample sizes and conflicting results have been inconclusive. We randomised 2405 primiparous low-risk women in spontaneous labour, before 6cm of cervical dilatation, into either water only group or light diet (at will.) The primary outcome was the spontaneous vaginal delivery rate (SVD). Duration of labour (DOL), instrumental delivery rate, caesarean section rate, induction of labour, incidence of vomiting, baby’s birth weight, Apgar scores and need for admission to SCBU/NICU were also evaluated. No significant difference was observed regarding SVD (44% vs. 45%; RR, 0.986; 95% CI 0.9–1.1), DOL (704 mins vs. 726 mins p=0.18), incidence of vomiting (`once’, 18% vs. 16%; RR, 1.09; 95% CI 0.91–1.3; p=0.36; `more than once’, 17% vs.17%; RR, 1.007; 95% CI 0.85–1.2; p=0.96) and neonatal endpoints between the two groups. Light dietary intake during labour did not influence labour outcome.

An alteration in the balance of apoptotic regulators Smac and XIAP in placentae of pregnancies complicated by pre-eclampsia

HR Buttle, I Crocker, P Baker, A Heazell

Maternal and Fetal Health Research Centre, St Mary’s Hospital, Manchester, M13 OJH

Apoptotic cell death, a potential factor in the pathogenesis of pre-eclampsia (PE) is regulated by a balance between pro- and anti-apoptotic proteins. We hypothesised that the expression of pro-apoptotic Smac and anti-apoptotic XIAP would be altered in placentae of pregnancies complicated by PE. The expression of Smac and XIAP were semi-quantified by Western blotting and localised using immunohistochemistry in placentae from normal pregnancies (n=8) and PE (n=9). The expression of Smac was increased in placentae in PE; this localised to the syncytiotrophoblast (ST) cytoplasm. XIAP also localised to the ST cytoplasm, but there was no altered expression in PE. The increased trophoblast apoptosis in PE is associated with an alteration in the balance of Smac and XIAP, providing further evidence of activation of cellular apoptotic pathways in PE.

Never the twain shall meet! Achievement of patient centred goals and objective outcome measures in pelvic floor dysfunction surgery

S Srikrishna, D Robinson, L Cardozo

Kings College Hospital, London

The aim of this study was to compare objective outcome measures with achievement of patient centred outcomes in women undergoing continence and pelvic reconstructive surgery. The secondary aim was to compare patient overall satisfaction with goal achievement and with that of the surgeon. This prospective longitudinal observational study recruited women on the waiting list for pelvic reconstructive or continence surgery from a tertiary referral centre. Pre-operative objective assessment included clinical examination (POP-Q) and videocystourethrography (VCU). Patients and surgeons listed up to 5 personal goals following surgery and documented degree of goal fulfilment at the at 6 weeks and 3 months post operative review. Women were also re examined and completed a Patient Global Impression of Improvement (PGII). SPSS (V 13 Chicago Illinois) was used for statistical analysis, with paired t- test (POP-Q) and Wilcoxon Signed Rank (PGII). 35 women were recruited over 4 months Objective assessment showed statistically significant improvement. (p<0.05) Mean goal achievement was 67% per patient and 83% per surgeon. PGII scores showed a significant improvement from the 6 week (1.68) to the 3 month review (1.37). This study confirms that patient centred goals are subjective. There is not always a correlation between objective and subjective “success”. Surgeon’s goals are more consistently similar, largely based on objective outcome measures and more significantly achieved.

Long-term comparative effect of hysterectomy and transcervical endometrial resection on urinary incontinence symptoms

S Allahdin, QA Warraich, K Harrild, C Bain

Aberdeen Royal Infirmary and University of Aberdeen

Total abdominal hysterectomy (TAH) can potentially disrupt the pelvic anatomy, nerve supply and increase the incidence of urinary incontinence symptoms (UIS).

Ethical approval was obtained for a retrospective case note review. Women who had a TAH or transcervical resection of the endometrium (TCRE) for dysfunctional uterine bleeding from 01/01/1990 to 31/12/1994, with no UIS before their operation, were included. Logistic regression was conducted to investigate associations with UIS.

316 women had a TAH and 229 a TCRE. Both groups were similar in demographic characteristics. Hospital referrals for UIS (P = 0.011), urological investigations (P= 0.021) and treatment of UIS (P=0.008) were higher in TAH group at 10 years follow up. After adjusting for some demographics, the TAH group were still more likely to be referred for UIS at 10 years, odds ratio 2.5, 95% CI (1.30, 4.84). TAH is associated with an increase in UIS at long term follow up.

Selecting failing pregnancies in the pregnancy of unknown location (PUL) population to reduce follow-up

E Kirk, L Tan, G Condous, B Van Calster, C Bottomley, S Van Huffel, D Timmerman, T Bourne

St George’s Hospital, University of London, Cranmer Terrace, London, SW17 0RE

The aim was to develop a strategy for the management of PULs based on the predicted outcome at 48hrs using a logistic regression model. Women classified with a PUL had serum hCG levels taken at 0 and 48hrs and were managed according to the model’s prediction. If the model predicted an intra-uterine pregnancy (IUP) or ectopic pregnancy (EP), women were rescanned on day 7; if the model predicted failing PUL and the hCG ratio (hCG 48hrs/hCG 0hrs) was also <0.87, these women were discharged; if the model predicted failing PUL and the hCG ratio was also >0.87, serum hCG was repeated on day 7. 363 PULs were managed according to the model’s prediction. 88.4% (321/363) had the location of their pregnancy confirmed in the time period according to the study and by day 7 only 3% (10) women were still PULs. A logistic regression model has been developed that can successfully predict the outcome of PULs and can be used as a basis on which to clinically manage such cases.

Arteriogenesis, uterine natural killer cells and recurrent miscarriage

H Nik,1 RG Farquharson,1 J Bulmer,2 G Lash,3 S Quenby4

1 Liverpool Women’s Hospital, Crown Street, Liverpool L8 7SS, UK; 2 School of Clinical and Laboratory Sciences and 3 School of Surgical and Reproductive Sciences, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE2 4HH, UK; 4 School of Reproductive and Developmental Medicine, University of Liverpool, Liverpool Women’s Hospital, Liverpool L8 7SS, UK

High numbers of uterine natural killer cells (uNK) cells have been associated with recurrent miscarriage but the mechanism by which they cause pregnancy loss is unknown. We aim to test the hypothesis that increased uNK cells are associated with disordered arteriogenesis. Forty six patients with three or more unexplained miscarriages were seen in the midluteal phase of their cycle and had uterine artery Doppler studies and an endometrial biopsy taken. Immunohistochemistry with antibodies to CD56, CD34, factor 8, smooth muscle actin and myosin was used to count uNK cell numbers and examine endothelial and smooth muscle differentiation in endometrial arterioles. High uNK cell numbers were associated with low uterine artery resistance (P=0.0004) and increased spiral arteriole smooth muscle differentiation. In conclusion we have preliminary evidence to suggest an association between high uNK cells and increased arteriogenesis potentially leading to excessive oxidative stress in early pregnancy.

Phenotypic characterization of the myometrium in normal and adenomyotic uteri

MK Mehasseb, MA Habiba, L Brown, SC Bell

Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester, LE2 7LX, UK

We studied the myometrium from normal (n=45) and adenomyotic (n=64) uteri in different phases of the cycle, we examined histological and Feulgen stained sections and used in-situ hybridization probes for mitochondrial RNA and snRNA. Semi-quantitative image analysis of sequential fields covering full myometrial thickness excluding the adenomyosis lesions revealed no distinct myometrial layering. The total nuclear area was higher at the Junctional Zone (JZ) compared to the subserosal myometrium in both normal (23.92±0.99 vs 15.15±0.68 mean ± SEM, p<0.05) and adenomyosis uteri (20.98±0.67 vs 13.29±0.55, p<0.05), with a gradient drop from the inner to outer myometrium. The nuclear area was lower in adenomyosis compared to normal JZ (p =0.013). The nuclear density at the JZ did not significantly vary with the cycle phase. Mitochondrial activity was higher in the subserosal myometrium, with no difference in proliferative activity. A hypodense JZ myometrium may play a role in the pathogenesis of adenomyosis.

Activation of IGF1R signalling pathways influences first trimester placental cell turnover

K Forbes, JD Aplin, PN Baker, M Westwood

University of Manchester, Division of Human Development, St Mary’s Hospital, Manchester

In the placenta abnormal regulation of proliferation and apoptosis have been implicated in pre-eclampsia, however the mechanisms by which these processes are regulated are poorly understood. We aimed to investigate how the IGF axis regulates trophoblast turnover First trimester tissue explants were cultured in serum free (SF) conditions at 20% oxygen for up to 4 days in the presence of BrdU (100μM) +/− IGF-I (10nM) or IGF–II (10nM), then proliferation (BrdU and Ki67 immunohistochemistry) and apoptosis (TUNEL) were assayed. IGF-I and -II enhanced levels of cytotrophoblast proliferation and decreased apoptosis. Signalling pathway inhibitors revealed that IGF-induced proliferation is mediated by IGF1R-mediated activation of MAPK whereas the survival effect is mediated by the PI3K pathway. Thus the IGF axis is an important regulator of placental cell turnover.

Correlating ultrasound placental grading with a stereological estimate of placental function

TT Yin,1 SS Ong,2 J Padfield,3 T Mayhew,4 P Loughna5

1 School of Human Development, Nottingham, 2 Birmingham Women’s Hospital, 3 Nottingham University Hospitals (QMC Campus), 4 School of Biomedical Science, University of Nottingham, 5 Nottingham University Hospitals (City Campus)

A high grade placenta on ultrasound is believed to reflect maturation. We wished to examine the relationship between early maturation and placental function. Stereological examination of placental tissue permits calculation of the oxygen diffusive capacity (Dp) as a surrogate for placental function. Placentas were graded by ultrasound at 31–34 weeks. After delivery, 32 placentas were collected and multistage systematic random sampling performed. Test point counting allowed estimation of the volume and surface area of functioning villi and fetal capillaries. The harmonic mean thickness (HMT) was obtained by measuring random intercept lengths. Dp was calculated from these measurements. Dp demonstrated a wide range (4.16–168.83 ml/min/kPa) with no significant difference between the four groups. There was no relationship between HMT and Dp. There is no correlation between Grannum grades and Dp. Early maturation of the placenta does not imply altered placental function as assessed by Dp and HMT.

Supplementation with vitamins C and E in pregnancy and liver function

L Manning, A Briley, L Poston, L Chappell, A Shennan

Maternal and Fetal Medicine Research Unit, 10th Floor North Wing, St Thomas’ Hospital, Lambeth Palace Road, SE1 7EH

Earlier this year a randomised controlled trial investigating the effects of antioxidant supplements in pregnancy reported higher levels of raised liver aminotransferase levels in subjects that received this intervention.1 Using information gleaned from the VIP trial2 database, the AST and ALT levels in subjects from this study are examined here to further investigate any relationship between liver function, disease severity, and supplementation with vitamin C and E in pregnancy. No significant difference in serum AST and ALT levels was observed between those taking the vitamin supplements and those taking a placebo in pregnancy. Of those subjects tested, women that were given the antioxidants had a mean AST of 30.74 IU/ml, versus a mean AST of 29.35 IU/ml in women that were allocated a placebo (p=0.8). Mean serum ALT levels were 30.74 IU/ml in the vitamin group and 27.53 IU/ml in the placebo group (p=0.846). In addition, it was found that there was no significant difference in the proportion of women with abnormal LFT results between the treatment groups. In the vitamin intervention group, 1.99% were known to have an AST level >50 IU/ml, and the corresponding percentage in the subjects taking a placebo was 1.72% (relative risk 1.088; 95% CI 0.624 to 1.895; p=0.766). 4.72% of those allocated the vitamin intervention had documented ALT levels >50 IU/ml, compared to 4.02% of the subjects given a placebo (relative risk 1.099; 95% CI 0.764 to 1.6; p=0.612). This was also observed when the results from women that had pre-eclampsia were examined alone. In conclusion we found no evidence to suggest that taking supplements of vitamin C and E during pregnancy either causes or exacerbates deranged liver function.

1. Rumbold A, Duley L, Crowther C, Haslam R, Dekker G, and Robinson J. 2006. Vitamins C and E and the risk of pre-eclampsia and perinatal complications. The New England Journal of Medicine; 354: 1796–806.

2. Poston L, Briley AL, Seed PT, Kelly FJ, and Shennan AH. 2006. Vitamin C and E in pregnant women at risk for pre-eclampsia (VIP trial): randomised placebo-controlled trial. Lancet; 367: 1145–54.

Histamine-stimulates calcium signalling in the ULTR human uterine smooth muscle cell via the H1 receptor

JM Willets, AH Taylor, JC Konje

Division of Reproductive Sciences, Department of Cancer Studies and Molecular Medicine, University of Leicester, Clinical Sciences Building, Leicester Royal Infirmary, Leicester, LE2 7LX

To examine the possible link between elevated maternal plasma histamine levels and pre-term labour, histamine receptor signalling was examined in ULTR cells, an immortalized human uterine smooth muscle cell line. ULTR cells were pre-loaded with 3μM of the calcium-sensitive dye Fluo4AM for 1 h before treatment with graded doses of histamine in the presence/absence of receptor antagonists. Alterations in intracellular Ca2+ ([Ca2+]i) concentrations were detected as increased cytosolic fluorescence using confocal imaging. Histamine produced a concentration-dependent increase in [Ca2+]i with an EC50 of 977 ± 50 nM, (n=48; mean ± sem) and was inhibited by the H1 antagonist, diphenhydramine (10μM), but not the H2 antagonist, cimetidine (10μM). Histamine-stimulated [Ca2+]i increases measured 5 min after an initial histamine challenge (100μM, 1 min), were reduced by 53.7 ± 4.1% and returned to baseline by 10 min (n=23). These data suggest that normal human myometrial histamine signalling may be mediated through H1 receptors.

Human labour is characterised by an inflammatory gene expression signal

SS Bollapragada, R Youssef, F Jordan, A Young, IA Greer, JE Norman, SM Nelson

Reproductive & Maternal Medicine, University of Glasgow, Third Floor, Queen Elizabeth Building, Glasgow Royal Infirmary, Glasgow G31 2ER

Human parturition is a coordinated process involving changes to myometrium, cervix and membranes. We have identified gene changes common to all three tissue types with human labour at term. Gene arrays (Affymetrix v2 U133+2) were carried out on myometrial and cervical biopsies (n=9) obtained from labouring and non-labouring women undergoing caesarean section at term. These were compared with the gene expression profile for membranes, available in the literature. 224 genes were differentially expressed by labour in membranes, of these, 122 were expressed by myometrium and 140 by cervix. There was a strong correlation between the genes expressed by the three tissues: membranes v myometrium (r=0.56, p<0.001), membranes v cervix (r=0.52, p<0.001) and myometrium v cervix (r= 0.64, p<0.001). 99 genes were common to all 3 tissues, however only 67 were consistently up-regulated and 17 down-regulated. Genes associated with inflammation and induction of chemotaxis demonstrated the greatest fold change in all three tissues and therefore labour appears to involve “a common inflammatory pathway”.

Natural antimicrobials and bacterial vaginosis in pregnancy

SJE Stock, L Duthie, RW Kelly, SC Riley, AA Calder

Centre for Reproductive Biology, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ

Natural antimicrobials are host defence proteins with antibacterial and antiviral properties, which can also modulate the adaptive immune response. We examined natural antimicrobial levels in cervicovaginal secretions of women in the second trimester of pregnancy (n=113) using enzyme linked immunosorbent assay. Bacterial vaginosis status of these women was determined by gram stain using Nugent’s criteria. We also used quantitative PCR to study natural antimicrobial production by vaginal and cervical cell lines. We found the natural antimicrobials human beta-defensin 2, secretory leucocyte protease inhibitor (SLPI) and elafin are produced by vaginal and cervical cells. Cells derived from vaginal epithelium produced greater amounts of natural antimicrobials than cervical cells in vitro. Significantly lower (p<0.05) SLPI levels were found in cervicovaginal secretions of women with bacterial vaginosis. Natural antimicrobials may be an important component of vaginal host defence, and altered production might contribute to the sequelae of bacterial vaginosis.

Cervical leukocyte sub-populations in idiopathic preterm labour

MK Whitworth, J Pafilis, G Vince, S Quenby

University of Liverpool Department of Reproductive and Developmental Medicine, Liverpool Women’s NHS Foundation Trust, Liverpool, L8 7SS, UK

Approximately 50% of premature births are the result of spontaneous preterm labour (PTL) of no known cause. It has been postulated that cervical ripening, which occurs in PTL, resembles an inflammatory response. We tested the hypothesis that cervical leukocytosis is associated with the onset of recurrent idiopathic preterm labour.

We developed a, non-traumatic method of sampling the cervix. A cytobrush was used to sample the epithelium at 12 and 20 weeks of the cervix of 160 women with a past history of idiopathic spontaneous PTL. Leukocyte sub-populations were examined using immunocytochemistry.

There was no significant longitudinal change or association between cervical leukocytes and ultrasound detected cervical shortening. Women with idiopathic preterm labour that recurred had fewer cervical macrophages at the beginning of the second trimester of pregnancy the those whose PTL did not recur (P<0.01).

Cervical epithelial macrophages appeared to prevent PTL recurring possibly by preventing ascending infection.

The inhibitory effect of Δ9-tetrahydrocannabinol on trophoblast cell proliferation and transcription is mediated via the CB-2 receptor

AH Taylor, MS Abbas, SC Bell, JC Konje

Reproductive Sciences Section, Department of Cancer Studies & Molecular Medicine, University of Leicester Medical School, Leicester, LE2 7LX, UK

Previously, we demonstrated that Δ9-tetrahydrocannabinol (THC), in physiologically relevant concentrations, inhibited the growth and tight transcriptional control of the BeWo trophoblast cell. We sought to find the receptor that mediated this effect and thus treated BeWo cells with up to 30μM THC in the presence and absence of the CB1-, and CB2-specific antagonists, SR141716A and SR144528. The results showed that the 40% reduction in cell number after 48hr treatment with 30μM THC was only abrogated with SR144528. These data suggested the presence of a functional CB2 but not a functional CB1 receptor in BeWo cells. RT-PCR with gene-specific primers confirmed the presence of transcripts for CB2 but not for the CB1, supporting this conclusion. Although THC caused a 1.7-fold increase in the levels of the transcriptional regulator HDAC3, only the CB2 antagonist prevented this effect, suggesting that THC-induced HDAC3 expression and inhibition of BeWo cell proliferation are causally linked via CB2.

Role of epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraeinoic acids (HETEs) in pregnant myometrium

T Pearson, D Barrett, R Khan

Centre for Reproduction and Early life, Institute of Clinical Research, University of Nottingham and Department of Obstetrics and Gynaecology, Derby City Hospital, Derby, UK, DE22 3DT

Epoxyeicosatrienoic and hydroxyeicosatetraeinoic acids are generated from arachidonic acid by the action of cytochrome P450 enzymes. The EETs and HETEs have effects on smooth muscle which increase or decrease contractility, respectively, and may therefore be a factor in the control and initiation of labour. Myometrial biopsies gathered from non-labouring women at elective caesarean section after informed written consent were utilised in these studies. Positive immunoreactivity to all P450 enzymes examined, CYP450 2C9/19, 2J2 (EET generating) and CYP450 4A, 4F3 (HETE generating) was found, particularly so for CYP450 4A. Western blotting showed clear signals for CYP450 2C9/19 and CYP450 4A. Isometric tension recordings from muscle biopsies, undertaken to determine the efficacy of 5,6-, 8,9-, 11,12-, and 14,15-EETs, showed relaxation to 5,6-EET. The balance between the concentrations of the EETs and HETEs may thus influence contractility and the labouring process. It has yet to be determined if P450 enzyme expression reflects altered myometrial activity.

Oxidative stress induces the vascular endothelial dysfunction of pre-eclampsia in vitro

S Moll, M Wareing PhD, M Sweeney PhD, P Baker DM FRCOG, I Crocker PhD

Division of Human Development, University of Manchester, United Kingdom, M13 0JH

Impaired endothelial dependent relaxation (IEDR) is believed to be a causative component of pre-eclampsia. As oxidative stress is implicated in pre-eclampsia, we hypothesised that chronic oxidative stress, in the form of exposure to hydrogen peroxide (H202), would induce IEDR in the maternal microvasculature. In this study, human myometrial arteries obtained from hysterectomy samples were incubated for 3 hours with H202 (0, 10, 50, 100μM) before functional assessment by wire myography. Additional myometrial biopsies were exposed to identical conditions and homogenised for the assessment of malondialdehyde (MDA), an indicator of oxidative stress and immunostained for 7,8-dihydro-8-oxo-guanine, an additional marker of oxidation. H202 at the highest dose (100μM) caused significant IEDR in myometrial vessels. This dose induced a marked increase in MDA and the appearance of 7,8-dihydro-8-oxo-guanine, localised to the microvascular endothelium. These results confirm the induction of oxidative stress by H2O2 and highlight its potential in inducing IEDR, a pathophysiological feature of pre-eclampsia.

Uterine artery doppler to predict fetal Growth Restriction: a systematic review and bivariate meta-analysis

JS Cnossen,a RK Morris,c BWJ Mol,b G ter Riet,a JAM van der Post,b PJE Bindels,a S Robson,d J Kleijnen,e A Coomarasamy,c KS Khanc

a Department of General Practice and b Department of Obstetrics and Gynaecology, Academic Medical Centre, Amsterdam, The Netherlands; c Department of Reproductive and Child Health, University of Birmingham, Birmingham Women’s Hospital, Birmingham, UK; d School of Surgical and Reproductive Sciences, University of Newcastle, Newcastle, UK; e Kleijnen Systematic Reviews Ltd, York, UK

Alterations in flow velocity waveforms measured in uterine arteries are associated with subsequent development of fetal growth restriction (FGR). We performed a systematic review to determine the accuracy of uterine artery Doppler indices in predicting FGR and to assess its potential clinical value.

Medline, Embase, Cochrane Library and MEDION were searched from inception to April 2006. We also searched reference lists and contacted experts. All studies reporting on uterine artery Doppler before the 25th (mean) gestational week with a measurement of FGR as outcome and allowing construction of a 2×2 table were included. Bivariate meta-analysis was performed. Sixty studies, testing 40,637 women were included. For FGR, resistance index with notching showed the best sROC curve (mean sensitivity 40% (95% CI 20–61); mean specificity 91% (95% CI 87–95). The best specificity was achieved by pulsatility index with notching (99%; sensitivity 12% (95% CI 8–18). We conclude that uterine artery Doppler has a limited role to play in the prediction of FGR.

Uterine effects of levonorgestrel-impregnated intrauterine device (LNG-IUS) inserted at the onset of tamoxifen treatment in post-menopausal women with breast cancer

R Panchal SC Bell and JC Konje, L Brown, MA Habiba

Department of Cancer and Molecular Medicine, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, PO Box 65, Leicester LE2 7LX, UK

We assessed whether insertion of the levonorgestrel-intrauterinedevice (LNG-IUS) at the onset of tamoxifen treatment for breast cancer could prevent uterine growth and the development of tamoxifen associated endometrial pathology. Postmenopausal women with breast cancer due to commence tamoxifen were randomised to either a LNG-IUS (n=16) or control group (n=19). Endometrial and uterine dimensions were measured by ultrasonography at 0 and 6 months, and endometrial biopsies taken at 0 and 12 months. Polyps were assessed by hysteroscopy. The transverse uterine dimension and uterine volume increased by 6.4% and 17.2% respectively in the control group, but decreased by 5.2% and 9.6% in the LNG-IUS group. At 12 months, the endometrium from the LNG-IUS group exhibited a pseudo-decidual response in 100% of cases. There were 3 new polyps in the control group and none in the LNG-IUS group at 12 months. The LNG-IUS counters the uterotrophic effects of tamoxifen and induces endometrial pseudo-decidualisation.

Positive predictive value of ultrasound for the diagnosis of molar pregnancy

E Kirk, AT Papageorghiou, L Tan, G Condous, C Bottomley, T Bourne

St George’s Hospital, University of London, Cranmer Terrace, London, SW17 0RE

Previous studies have examined ultrasound findings in histopathologically confirmed cases of gestational trophoblastic disease (GTD). As a consequence the positive predictive value (PPV) of ultrasound for diagnosis is overestimated. The aim of this study was to assess the ultrasonographic findings in all women suspected of having GTD on ultrasound and those with histologically confirmed GTD, in order to obtain a true sensitivity and PPV for ultrasound diagnosis. A retrospective analysis was performed of all cases presenting to the Early Pregnancy Unit over a four-year period - 90 cases. In total, 61 women had GTD confirmed on histology - 41 partial hydatidiform moles (PHM) (67%) and 20 complete hydatidiform moles (CHM) (33%). The overall sensitivity and PPV for the ultrasound diagnosis of GTD was 44% and 48% respectively. For PHMs the respective values were 20% and 22% and for CHMs they were 95% and 40%. Ultrasonography is more reliable for diagnosing CHMs rather than PHMs. Overall the sensitivity of ultrasound for accurately predicting MP is 44%, and one in two women with an abnormal scan will have the disease confirmed on histology.

Disordered myometrial organisation during early uterine development in tamoxifen-induced adenomyosis

MK Mehasseb, SC Bell, INH White, MA Habiba

Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester, LE2 7LX, UK

We studied early uterine development in female CD-1 pups, treated with oral Tamoxifen (1mg/kg), from age 1 to 5 days as a model for adenomyosis. Uteri from treated (n=25) and control (n=25) mice were obtained on days 2, 6, 10 and 15 of age. We examined sections histologically, using image analysis and immunohistochemistry for the muscle diffentiation markers; alpha-actin, desmin, and vimentin. Undifferentiated Mullerian mesenchymal cells developed to form the endometrial stroma, and the inner circular (alpha-actin weakly positive day 6, strongly positive day 10) and outer longitudinal (alpha-actin positive day 15) myometrium. Following tamoxifen exposure, uteri were heavier due to oedema on day 5, but significantly smaller thereafter. Early signs of adenomyosis were observed on day 10. The inner myometrium showed disruption with lack of continuity, disorganisation and bundling, allowing endometrial invasion deep in the myometrium. Disruption of the inner myometrium may play a role in the development of adenomyosis.

A comparison of the IVF / ICSI (in vitro fertilisation/intracytoplasmic sperm injection) performance of women of Asian origin with white Caucasians

T Girish, R Neuberg, AC Davidson, J Blower

Leicester Fertility Centre, University Hospitals of Leicester NHS Trust, Leicester, LE1 5WW

Of the many factors affecting the outcome of IVF/ICSI treatment, the least studied aspect is ethnicity. A large proportion of the published literature on assisted reproduction relates to white women, and treatment regimes are usually optimised to suit responses in this ethnic group. Our objective was to compare the reproductive performance of women of Asian origin with white Caucasians in fresh IVF/ICSI cycles. Methods: A retrospective analysis of 248 fresh cycles of IVF/ICSI; 190 white Caucasians and 58 Asians, all of whom received treatment in the year 2005. Primary outcome measures: Clinical pregnancy rate and rates of abandoned cycles (due to poor and over-response). Secondary outcome measures: Total dose and days of gonadotrophins required, number of follicles >14 mm size, number of eggs retrieved, fertilised, cleaved, transferred, frozen, grading of embryos and risk of OHSS. Clinical pregnancy rate/cycle initiated was higher in Caucasians: 26.8% versus 13.8%. The rate of abandoned cycles was higher in Asians: 25.8% versus 15.2 %. There was no significant difference in any of the secondary outcome measures. Under the same IVF/ICSI regime, Asians performed worse than white Caucasians in terms of clinical pregnancy rate and had higher rate of abandoned cycles at the stage of ovarian stimulation. We need large randomised controlled trials comparing the reproductive performance of Asians with white Caucasians.

What is the clinical utility of cystoscopy and bladder biopsy for lower urinary tract symptoms (LUTS)?

R Cartwright, J Bidmead, L Cardozo, D Robinson, M Vella, E Stasiowska

King’s College Hospital London

We prospectively studied 60 consecutive women referred for cystoscopy with LUTS. The presumptive diagnosis and preferred management plan were declared in advance of the procedure. At 6-week follow-up, an independent urogynaecologist reviewed all cases, to evaluate the contribution of cystoscopy and biopsy to diagnosis and management, and to assess the rate of complications. The cystoscopic appearance was entirely normal in 29 cases (48.3%). 18 (30.0%) biopsies made a positive diagnosis. There were no cases of carcinoma identified. The overall diagnostic yield was 43.3%. 11 cases (18.3%) had a new unsuspected diagnosis established. Overall the management was changed in 12 cases (20.0%). There were 3 (5%) complications noted, including two cases of frank haematuria, and one UTI. We conclude that cystoscopy and bladder biopsy are an important part of the investigation of LUTS. Together they have a high diagnostic yield, and change the management in a substantial proportion of cases.

The role of fetal macrophages in placental development

VJK Cookson, KA Ingman, JD Aplin

Division of Human Development, Medical School, The University of Manchester

Hofbauer cells are the resident tissue macrophages of the placental villous core. Unlike other macrophages their principal role in early pregnancy may not be induction of a classical immune response, as they lack HLA-DR. We hypothesised that Hofbauer cells interact with other resident cell populations in the villous core to achieve placental morphogenesis and angiogenesis. CD68+/MPA+/CD163+ Hofbauer cells were localised in first and second trimester placenta close to immature blood vessels and within collagen-free stromal channels. Methods were developed for the isolation, culture and characterisation of mixed stromal cell populations. Hofbauer cells retained their in vivo phenotype, were highly motile and survived for several weeks in coculture. The cultures digested collagen gels, and conditioned medium was shown to stimulate tubule formation by endothelial cells (HUVEC). In conclusion, stromal, and in particular Hofbauer cells, have important roles in angiogenesis and extracellular matrix remodelling within the developing placenta.