Abstracts presented at the competition meeting of the Blair Bell Research Society on the 4–5th December 2003 at the Royal College Obstetricians and Gynaecologists, London

Enhorning's theory of urinary continence is it correct? C. Chaliha, L. Yeo, P. Rahmanou and V. Khullar. Urogynaecology Unit, Department of Obstetrics and Gynaecology, St Mary's Hospital, Imperial College, London.

Enhorning's theory has proposes that urinary continence occurs when increases in intra-abdominal pressure are equal to the urethra and bladder. Incontinence occurs when the pressure increases are not transmitted to the urethra. This study determines whether urethral pressure increases during coughing are due to the urethral sphincter and are not positional.Women were recruited and underwent urethral pressure profilometry with a microtip catheter. A 3 D transperineal urethral ultrasound scan was then performed to calculate rhabdosphincter and urethral volumes. 19 women underwent both tests. The difference between the intra-urethral and intra-vesical pressures (Pure-Pves) were calculated for each cough and averaged for each quartile of the urethra and correlated with rhabdosphincter volume for each quartile.

A significant good correlation {r > 0.7, p < 0.05} was found between the volume of the sphincter and Pure-Pves only in the continent women (n = 10). In conclusion Enhorning's theory is incorrect and Pure-Pves during a cough is related to the size of the urethral sphincter.

Sexual function and genital sensation following feminising genitoplasty. Naomi Crouch,* Catherine Minto,* Gerard Conway,§ Lih-Mei Liao,* Christopher Woodhouse, Sarah Creighton.**Department of Gynaecology, Elizabeth Garrett Anderson Hospital, UCLH. §Department of Endocrinology, The Middlesex Hospital, Institute of Urology, The Middlesex Hospital.

The standard practice of feminising genitoplasty surgery for children born with ambiguous genitalia remains highly controversial, with little known regarding long-term outcomes. We recruited five adult women with 21-OH Congenital Adrenal Hyperplasia (CAH) who had undergone feminising surgery in childhood, and five normal women as controls. Subjects were asked to complete a specialised sexual function questionnaire. Thermal, vibratory and light touch sensory thresholds were assessed to the clitoris using a GenitoSensory Analyzer (GSA Medoc Ltd), and Von Frey Filaments. Results showed a difference between the two groups for vibration sensation (p = 0.11) and light touch (p = 0.08), and was significant for sensation to warmth (p = 0.04), and to cold (p = 0.017). A significant difference was shown in global sexual difficulties (p = 0.024), and notably with anorgasmia (p = 0.015).

These striking findings show that clitoral sensation and sexual function is markedly impaired in women who have had feminising surgery, and must be evaluated further in the light of the current contentious debate.

InhibinA as a predictor of pregnancy outcome in recurrent miscarriage. A. Prakash,1 E. M. Tukermann,2 T. C. Li,2 S. M. Laird,3 W. L. Ledger.11Department of Reproductive Medicine, University of Sheffield, 2Biomedical Research Unit, Jessop Wing, 3Division of Biomedical Sciences, Sheffield Hallam University, City Campus.

Recurrent miscarriage is defined as three or more consecutive miscarriages. A biochemical marker that may allow prediction of pregnancy outcome in such patients would be clinically useful. Inhibin may be one such biochemical marker useful for predicting viability of pregnancy. Inhibin is a glycoprotein with two active forms, Inhibin A and Inhibin B. Serum concentrations of Inhibin A rise rapidly in early pregnancy, whereas Inhibin B is undetectable at this time. We retrospectively analyzed serum sample from 20 women with recurrent miscarriage who had conceived spontaneously. Serum levels of Inhibin A were measured twice in the first trimester. These levels were correlated with pregnancy outcome in terms of miscarriage, preterm labour or term pregnancy. Inhibin A levels rose at least two fold in all the pregnancies. However this rise was greater in pregnancies going to term as compared to those with preterm labour or miscarriage. This preliminary study shows a possible role of Inhibin A as a marker for predicting pregnancy outcome.

Raised blood pressure in second generation offspring of protein restricted rat dams. C. Torrens, L. Poston* and M. A. Hanson. DOHaD Centre, University of Southampton, SO16 5YA. *MFRU, Guy's, King's and St Thomas' Hospitals, London, SE1 7EH. (c.torrens@soton.ac.uk).

Protein restriction during pregnancy results in raised blood pressure and endothelial dysfunction in the F1 offspring, but can such effects be passed to the F2 generation?

Systolic blood pressure (SBP) of F2 rats from F1 offspring of control (C; 18% casein) and protein restricted (PR; 9% casein) dams was recorded by tail cuff plethysmography at ca. 120 days of age.

F2 offspring from either a male or female PR F1 parent had raised SBP compared to F2 where both parents were controls.

Thus we show for the first time that protein restriction during pregnancy initiates cardiovascular effects passed to the grandchildren via both male and female lineages.

Long-term consequences of pregnancy: the persistence of fetal cells in maternal organs. Keelin O'Donoghue,1 Jerry Chan,1 Josu de la Fuente,2 Nigel Kennea,1 Jonathan R. Anderson,3 Ann Sandison,4 Irene A. G. Roberts2 and Nicholas M. Fisk.11Institute of Reproductive and Developmental Biology, Imperial College London, 2Department of Haematology, Division of Investigative Sciences, Imperial College London, 3Department of Cardiothoracic Surgery, Hammersmith Campus, 4Department of Histopathology, Charing Cross Hospital.

Fetal cells enter the maternal circulation during pregnancy and persist in women with autoimmune disease. However, the frequency of fetomaternal microchimerism and its cell type is unknown. We identified fetal mesenchymal stem cells (fMSC) in fetal but rarely in maternal blood. To test the hypothesis that fMSC engraft maternal organs, we obtained bone marrow and rib sections from women undergoing sternotomy/thoracotomy. Marrow cells were cytospun and cultured for MSC and XYFISH and immunostaining performed on cytospins and paraffin-embedded rib sections. Male cells were identified in marrow and MSC cultures from women with male pregnancies, but not in controls. MSC were characterized morphologically, immunophenotypically and by multilineage differentiation. Male cells were also identified in ribs from women with sons. fMSC engraft bone marrow after pregnancy and remain microchimeric for decades. This has implications for obstetric complications increasing fetomaternal trafficking, for graft survival and for gender and parity differences in disease predisposition.

Allelic carriage of the interleukin 13–1055 (C/T) functional single nucleotide polymorphism determines susceptibility to very preterm labour (<32 weeks). H. L. Logghe, N. Mistry, N. A. B. Simpson, M. I. Levene, N. M. Orsi, J. J. Walker. Academic Unit of Paediatrics, Obstetrics and Gynaecology, Leeds General Infirmary, Leeds.

Preterm labour (PTL) results from an inflammatory process. We hypothesised that an increased profile of the anti-inflammatory cytokine interleukin (IL)-13 could be protective. A functional IL-13 (C→T) single nucleotide polymorphism has been described, the variant T allele confers a high-secretory phenotype. This study examined the association between maternal IL-13 genotype and PTL, with or without premature rupture of fetal membranes (PROM). The preterm group included pregnancies <32 weeks' gestation with/without PROM (n = 70), controls had term deliveries (n = 71). Allele frequency of the variant T allele was significantly lower in the PTL group compared to controls (0.12 vs. 0.22; P < 0.05; OR 0.49; CI 0.26–0.94), particularly in the PROM subgroup (0.08; P < 0.05; OR 0.32; CI 0.13–0.80). Thus, carriage of the high-secretory phenotype T allele may reduce susceptibility to PTL and PROM. Furthermore, IL-13 SNP genotype could be used as a genetic screening marker for susceptibility to PTL.

Learning from adverse events: identification and assessment of severe maternal morbidity. V. Brace,1,2 M. Hall,2 G. Penney.1,21Scottish Programme for Clinical Effectiveness in Reproductive Health. 2Department of Obstetrics and Gynaecology, University of Aberdeen.

We aimed to quantify severe maternal morbidity throughout Scotland and to develop a system for assessing clinical management for the principal category-major obstetric haemorrhage. Fourteen inclusion events were defined. A structured proforma was developed for local assessment of management of obstetric haemorrhage. All 22 consultant-led maternity units participated. Events were reported monthly. For haemorrhage events, proformas were collated for anonymous national reporting. Between January and June 2003, 198 events were reported in 146 patients (5.8 patients per 1000 deliveries). Major obstetric haemorrhage accounted for 66% of cases. Completed proformas were received for 81 of the 96 reported haemorrhages (84%). Of these, 40% were associated with emergency caesarean section and 45% were attributed to uterine atony. Most haemorrhages were managed appropriately. However, the need to involve senior staff, especially consultant anaesthetists, early was a recurring issue. Combining local assessment with national collation and reporting provides the widest educational benefit.

Cystic fibrosis carriage—a concern in azoospermic males? E. V. Mocanu, R. Shattock, D. Barton, M. Rogers, O. Sheils, J. J. O'Leary, R. F. Harrison. HARI Unit, Rotunda Hospital, Dublin 1, Coombe Women's Hospital and St James's Hospital, Dublin 8, National Centre for Medical Genetics, Our Lady's Hospital for Sick Children, Crumlin, Dublin, Royal College of Surgeons in Ireland, Dublin 1.

It is not clear from the published literature if subfertile males have a higher carrier rate of CFTR gene mutations compared with their fertile counterparts. Blood DNA from 116 fertile men and 82 azoospermic males (abnormal karyotypes, Y deleted and CBAVD excluded) requiring ICSI were screened for 31 CF mutations. In the azospermic group 14 patients carried CF mutations (17.1%), while in the fertile group only 9 (7.7%). This study shows a significantly higher carrier rate of CFTR gene mutations in azoospemic males when compared with fertile males and suggests routine screening of all azoospermic males for CF carriage.

Investigating the effect of pre-eclamptic plasma on microvessel permeability in-vivo. Alyson Hunter,1 Chris Neal,2 Steve Harper,2 Peter Soothill,1 Dave Bates.21Division of Obstetrics and Gynaecology, St Michael's Hospital, University of Bristol, UK. 2Microvascular and Cardiovascular Research Laboratories, Department of Physiology, University of Bristol, UK.

Pre-eclampsia (PET), caused by endothelial dysfunction, results in increased vascular permeability. Plasma was collected from women with severe PET (n = 6), mild PET (n = 10) and normotensive controls (n = 45). The samples were then perfused through frog mesenteric microvessels to investigate their effects on the vessel permeability. The hydraulic conductivity (Lp) was measured for each sample across the microvessel membrane using a modification of the Landis technique. Perfusion of plasma from patients with severe PET resulted in a rapid transient increase in Lp from 1.6 ± 0.26 to 11.5 ± 2.3 × 10−7 cm.s−1.cmH2O−1 (p < 0.02) This effect was not seen in either the mild PET or control group. These results show that a macromolecule in pre-eclamptic plasma can increase microvascular permeability. VEGF is a macromolecule that has been implicated in pre-eclampsia. In the plasma used in the above experiments VEGF was found to be significantly increased in the PET women (median = 188 pg/ml) compared to the controls (median = 68 pg/ml) (p < 0.001).

Does labouring human myometrium differ from non-labouring? Susan Joanne Pierce,1,* Ebtsam Monir-Bishty,1 Siobhan Quenby* and Susan Wray.11Physiology and *Obstetrics and Gynaecology, The University of Liverpool, Liverpool, United Kingdom, L69 3BX.

Clinically the difference between labouring and non-labouring myometrium is enormous. However, it is unclear how much intrinsic difference there is and indeed, how similar labouring tissues are to each other? In this first report comparing them, the major findings are: Surprisingly, in vitro, there was no difference in frequency, duration or amplitude of contractions and Ca2+ transients between the two groups (n = 18, t-test: p < 0.05). Thus in vitro, factors controlling excitability and contraction are similar, irrespective of tissue state.Acidification and hypoxia were potent depressors of force and Ca2+, however a subgroup of labouring tissues showed increased resistance, indicating metabolic differences. Clinical records revealed that this subgroup was labouring efficiently (caesarean performed for other indications), whereas those performed for failure to progress succumbed to acidification and hypoxia. This subgroup also had significantly lower myometrial blood pH (7.35 ± 0.02) than those labouring efficiently (7.48 ± 0.02). Thus the answer to the question is yes and no!

Fenofibrate-mediated inhibition of endometrial cancer cell growth occurs in association with activation of the PPAR-alpha receptor. Samir A. Saidi, Cathrine M. Holland, D. Stephen Charnock-Jones, Stephen K. Smith. Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge.

We have previously shown that the PPAR-alpha agonist fenofibrate inhibits proliferation and induces apoptosis in endometrial cancer cell lines. We wished to determine whether this effect is mediated via PPAR-alpha and investigate the effects of agonists of the related PPAR-gamma and Retinoic Acid Receptor (RXR) receptors. Cell proliferation assays and FACS analysis were used to assess proliferation and apoptosis in Ishikawa endometrial cancer cells. We used a luciferase reporter assay system to demonstrate activation of the PPAR pathway. The effects of fenofibrate on apoptosis and cell proliferation were not observed with PPAR-gamma agonists. A significant increase in PPAR gene reporter activity confirmed activation of the PPAR receptor. Enhanced effects were seen when fenofibrate was used in combination with 9-cis retinoic acid. Fenofibrate inhibits growth of Ishikawa endometrial cancer cells in association with PPAR-alpha activation and shows therapeutic potential in endometrial cancer, particularly in combination with retinoic acid.

The effect of mechanical stretch on NFkappaB DNA binding in human amnion. Aarthi R. Mohan, BSc MB BS, Suren R. Sooranna, PhD, Yun S. Lee, PhD, Phillip R. Bennett, MD PhD FRCOG and Mark Johnson, MD PhD MRCOG.

Stretch plays a role in labour. Stretching amnion increases COX-2 and PGE2 production. COX-2 is regulated by NFkappaB, which increases with labour. NFkappaB may represent a therapeutic target in preterm labour. This study showed that IL-1b, but not stretch increased AP-1 binding in amnion cells. IL-1b or static stretch increased NFkappaB DNA binding. Stretch and IL-1b caused a greater increase than either alone. A proteosome inhibitor, MG132 inhibited the effect of IL-1b but not of stretch. An IKK inhibitor inhibited both effects. Stretching myocytes activated AP-1 but not NFkappaB. We conclude that stretching amnion activates NFkappaB. Since activation is not inhibited by MG132 it is not through the classic pathway (IKK induced degradation of IkappaB). Inhibition of stretch and IL-1b induced NFkappaB by the IKK inhibitor may be because IKK also phosphorylates p65/p50. IL-1b and stretch activate NFkappaB by different mechanisms. Stretch has different effects upon amnion and myometrium.

Obstetric Anal Sphincter Injury—Does the repair technique affects the outcome? Ruwan J. Fernando,1 Abdul H. Sultan,2 Christine Kettle,1 Simon Radley,3 PMS O'Brien.11University Hospital of North Staffordshire, Stoke on Trent. 2Mayday University Hospital, Croydon. 3Queen Elizabeth Hospital, Birmingham.

Obstetric Anal Sphincter Injury (OASI) is a major cause of maternal morbidity following vaginal delivery. Evidence in literature in the management of OASI is limited and inconclusive. A prospective randomised controlled trial was designed to compare overlap and end-to-end repair techniques for OASI in preventing maternal morbidity. Sixty-four patients with OASI were randomised and followed up to twelve months with questionnaires, endoanal scans and anorectal manometry. There were no statistically significant difference between two techniques in terms of perineal pain, sexual function, and quality of life up to twelve months. At twelve months 24% complained of faecal urgency and urge incontinence in the end-to-end group whereas none complained in the overlap group (p = 0.006). There was no difference in the anorectal manometry and endoanal scan findings. These results suggest that overlap technique is associated with a significantly lower rate of faecal urgency and incontinence and therefore justifies a larger multicentre trial.

Proteomic analysis of the myometrial proteome: Signal transduction of the oxytocin receptor. N. C. J. de Wit, MSc,1,2 A. J. R. Heck, PhD2 and S. Thornton, DM.11Biomedical Research Institute, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom. 2Biomolecular Mass Spectrometry, Bijvoet Centre for Biomolecular Research & Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

Coordinated myometrial contractions during labour result in delivery. Oxytocin and its receptor play a fundamental role but the underlying cellular mechanism is poorly understood. We have used functional proteomics to elucidate the mechanism of human myometrial oxytocin receptor signal transduction.

Strips of human myometrium mounted in an organ bath under 2g tension were exposed to oxytocin, a selective oxytocin receptor antagonist or vehicle control. Following treatment, tissue was frozen in liquid nitrogen. Proteins were extracted, separated by 2D-gel electrophoresis and visualised by silver staining. Each protein extract was analysed in triplicate producing nine gels from each biopsy.

We identified more than 35 consistent changes in protein expression (n = 5 patients). Many proteins demonstrated increased expression in the presence of the oxytocin antagonist with a corresponding decrease with oxytocin. In conclusion, we have demonstrated a number of novel human myometrial proteins likely to be involved in oxytocin receptor signal transduction.

Correction of cord serum bile acid profiles after treatment of obstetric cholestasis with ursodeoxycholic acid. Dann A. T.,1,* Kenyon A. P.,1,* Seed P. T.,1,* Mallet A. I.,2,* Shennan A. H.1,* and Tribe R. M.1,*1MFRU, GKT Sch. of Medicine, London; 2Sch. of Science, University of Greenwich, Kent.

Background: Perinatal mortality and morbidity in obstetric cholestasis (OC) may be associated with an accumulation of bile acids (BA) in the fetal compartment.

Objective: To determine whether treatment of maternal OC with ursodeoxycholic acid (UDCA) alters cord serum BA profiles.

Methods: BA profiles were measured by HPLC-MS in mixed cord serum from gestation matched women with i) OC, n = 42 and ii) OC treated with UDCA, n = 25.

Results: UDCA significantly lowered total BA by 61% in the treated versus the untreated OC group (CI: 45–83%, p < 0.01), predominantly by reducing taurocholic acid (31% CI: 19–50%, P < 0.001) and glycocholic acid (49%, CI: 31–78%, p < 0.01).

Conclusion: Maternal UDCA treatment corrected the abnormal BA profiles in cord serum from OC pregnancies. UDCA could be a useful intervention to limit adverse fetal events in OC.

Alterations in the levels of the endocannabinoid, anandamide, during gestation—a potential role in pregnancy maintenance. Osama M. H. Habayeb,1 Anthony H. Taylor,1 Mark D. Evans,2 Marcus S. Cooke,2 David J. Taylor,1 Stephen C. Bell,1 Justin C. Konje.1 Reproductive Sciences1 and Genome Instability2 Sections, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK.

Anandamide, an endocannabinoid and endogenous ligand for the cannabinoid receptors CB1 and CB2, is a neurotransmitter. Recently however a role in early pregnancy has been proposed. We developed a robust HPLC/MS method to measure anandamide levels in plasma and determined whether levels alter during the menstrual cycle and pregnancy. Mean levels of anandamide during the follicular phase of the menstrual cycle were significantly higher than those in the luteal phase (1.68 ± 0.48 vs. 0.87 ± 0.54 nM mean ± SD; p < 0.01). Levels in the first trimester were similar to those in the luteal phase (0.88 ± 0.44 nM) but were lower during the second and third trimesters (0.44 ± 0.39 and 0.43 ± 0.18 nM respectively). At term levels in labour were significantly higher than those in women who were not in labour (2.52 ± 1.07 vs. 0.68 ± 0.44 nM; p < 0.001). These studies suggest that peripheral anandamide levels are hormonally regulated and that anandamide might play an important role during pregnancy and labour.

Normal human pregnancy is associated with an elevation in the immune suppressive CD4+/ CD25+regulatory T cell subset. D. A. Somerset,1 Y. Zheng,2 M. D. Kilby,1 D. M. Sansom,2 M. Drayson.31Department of Fetal Medicine, Division of Reproduction and Child Health, University of Birmingham, Birmingham Women's Hospital, 2MRC Center for Immune Regulation, University of Birmingham, 3Department of Immunology, Division of Immunity and Infection, University of Birmingham.

CD4+/CD25+ T regulatory (TReg) cells suppress auto-immune disease and allograft rejection in mice. Cells of similar phenotype and in vitro functional activity are present in normal human blood throughout life but their importance in vivo is difficult to ascertain. Pregnancy is associated with substantial changes in immune regulation that develop and abate over a programmed period of time; study of these changes might improve our understanding of TReg cell physiology. TReg cells were assessed in blood of 25 non-pregnant, 63 pregnant and 7 post-natal healthy women by flow-cytometry. There was a rapid increase in circulating TReg cells during early pregnancy, peaking during the second trimester and then a rapid decline post-partum. Isolated CD4+/CD25+ cells expressed a genetic marker of TReg cells-Foxp3. The proliferative response of autologous CD4+/CD25 T cells to allogenic dendritic cells was enhanced by the removal of these TReg cells and inhibited by their re-addition.

Endothelial and vascular smooth muscle function in a rat model for polycystic ovary syndrome. K. Lakhani,1 W. Yang,3 A. Dooley,4 E. El-Mahdi,5 M. Sundaresan,2 R. Bruckdorfer,4 A. Leonard,5 A. Seifalian,3 P. Hardiman.51Ultrasound Department and 2Department of Histopathology, North Middlesex Hospital, London; 3University Department of Surgery, 4Department of Biochemistry and Molecular Biology and 5Academic Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, The Royal Free Hospital, London.

Changes in isometric tension induced by acetylcholine (ACh) +/−100 μM L-NAME, and sodium nitroprusside (SNP), were assessed in phenylephrine precontracted aortic rings from a mifipristone-induced rat model of polycystic ovary syndrome (PCOS).

ACh-induced relaxation was reduced in PCOS aorta vs controls (68.0%vs 84.0%, 10 μM ACh, p = 0.001). L-NAME inhibited ACh-induced relaxation in PCOS aorta to a lesser extent than in controls (32.5%vs 11.0%, 10 μM ACh, p = 0.001). SNP induced aortic relaxation, to the same extent as ACh in controls, but to a greater extent in PCOS aorta. Indeed, SNP induced relaxation in PCOS aorta was greater than in controls (104%vs 87%, 1 μM SNP, p = 0.05).

These results suggest that nitric oxide dependent and independent mechanisms regulating aortic relaxation are disturbed in this PCOS model. Such changes may be relevent to the vascular dysfunction seen in PCOS women, and may be related to the endocrine disturbances described in this model.

Novel PgF receptor antagonism and BKCa channel activity in human uterus during pregnancy and preterm labour. Sexton D. J., Doheny H. C., O'Reilly M. W., Friel A. M., Morrison J. J. Department of Obstetrics and Gynaecology, Clinical Science Institute, University College Hospital Galway, National University of Ireland Galway.

PgF plays a pivotal role in parturition and exerts a potent stimulatory effect on the myometrium. THG113 has recently been reported as a novel potent PgF receptor (FP) antagonist. BKCa is central to myometrial excitability. There are no data in relation to the effects or secondary mechanism of action of THG113 in human myometrium. The aims of this study were to investigate the effects of THG113 in vitro, and on BKCa activity in single channel recordings, in dispersed human myometrial cells, using the patch clamp technique. Myometrial biopsies were obtained at caesarean section. THG113 exerted a potent relaxant effect on human uterine contractions (mean maximal inhibition 45.64%± 3.55; n = 6; P < 0.001), an effect which was antagonized by iberiotoxin (IbTX)(0.1 μM). In single channel recordings THG113 significantly increased the open state probability of the BKCa channels (control 0.023; 50 μM THG113 0.1356; P = 0.001; n = 6). Pre-incubation with IbTX similarly attenuated this effect. FP antagonism exerts a potent uterorelaxant effect, which appears to be mediated via BKCa with implications for preterm labour and tocolysis.

In pre-eclampsia (PE), circulating factor(s) precede clinical disease, irrespective of mid-trimester Doppler waveform and development of associated IUGR. Jenny E. Myers, BM BS,1 Maureen Macleod,2 Gary J. Mires, MD2 and Philip N. Baker DM.11Maternal and Fetal Health Research Centre, University of Manchester, Manchester, United Kingdom, M13 0JH and 2Maternal and Child Health Sciences, Ninewells Hospital and Medical School, Dundee, United Kingdom, DD1 4HN.

We studied circulating factor(s) in PE by assessing the effect of plasma on endothelial-dependent relaxation, using wire myography. Myometrial vessels from normal pregnant women were incubated with plasma samples (22 and 26 weeks) from women (n = 85) deemed at high/low risk of developing PE/IUGR on the basis of 22 week uterine artery Doppler studies. 19 women developed PE; 11/19 had associated IUGR. A further 19 women developed IUGR alone.Vessels incubated with plasma from women who subsequently developed PE, exhibited significantly attenuated endothelium-dependent relaxation. There was no effect of plasma from women with abnormal Doppler waveforms or with IUGR alone; likewise the additional development of IUGR did not alter the effect of plasma from women who developed PE. The circulating factor(s) effect was demonstrable up to 16 weeks before clinical disease and was specific to human vessels: plasma from women with established PE had no effect on endothelium-dependent relaxation of mouse mesenteric or uterine vessels.

Increased L-Cystine transport in Peripheral Blood Mononuclear Cells (PBM) and oxidative stress in pre-eclampsia. N. McCord,1 P. T. Y. Ayuk,1 I. L. Sargent,1 C. W. G. Redman1 and C. A. R. Boyd.2 Nuffield Department of Obstetrics and Gynaecology1 and Department of Human Anatomy and Genetics,2 University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom.

Pre-eclampsia is a state of systemic oxidative stress. Glutathione (GSH) is the major intracellular anti-oxidant. The rate-limiting step of its production is the transport of L-cystine across the cell membrane. Three main transport systems are involved, including system xc, system b0+ and system XAG. We aimed to determine whether normal pregnancy and pre-eclampsia are associated with activated transport systems for L-cystine and if this causes altered GSH levels in whole blood. Total L-cystine transport by PBM was up-regulated in pre-eclampsia but not in normal pregnancy. Real time RT-PCR confirmed the functional data and suggested that, specifically, system b0+ was up-regulated with increased expression of rBAT (heavy chain of system b0+). However, whole blood GSH was not increased, whereas the GSH/GSSG ratio was decreased, which is consistent with oxidative stress. Understanding the mechanisms and pathways involved in oxidative stress may lead to therapeutic interventions to treat or prevent pre-eclampsia.

Phytoestrogens reduce sperm motility in vivo and in vitro. L. Anderson,1 N. McClure1 M. Morton2 and S. E. M. Lewis1 School of Medicine, Obstetrics and Gynaecology, Queen's University Belfast.1 Willowbrook labs, St Mellons Cardiff.2

Genistein, a dietary phytoestrogen, is increasingly present in the male diet through soya substitution. Its endogenous effect on male development and sperm function is not understood.

In a series of 24 human subjects we have demonstrated genistein levels to be significantly higher in seminal fluid than venous blood suggesting a concentrated exposure for sperm to genistein. There was an inverse correlation between seminal genistein concentration and % sperm motility (r = 0.46; p < 0.05).

Using equivalent dietary genistein supplementation in a rat model over two generations, we established a consistent decrease in Sertoli cell numbers and an associated rise in serum FSH levels. Although there was a decreasing trend in litter numbers this was not significant.

These findings suggest that dietary genistein is associated with impaired sperm function and that developmental exposure may alter fertility.

Protein Kinase A catalytic and regulatory holoenzyme expression, activity and association with A kinase anchoring proteins in human myometrium during pregnancy and labour. M. MacDougall, G. N. Europe-Finner, S. C. Robson. School of Surgical and Reproductive Sciences, Newcastle University, Newcastle-upon-Tyne.

Cyclic AMP is a smooth muscle relaxant. Several components of the cAMP-signaling pathway are differentially expressed in the human myometrium during pregnancy. The ubiquitous cAMP signal is targeted through the use of differing catalytic and regulatory isoforms of PKA, and by PKA's association with A-kinase anchoring proteins (AKAPs), allowing cellular compartmentalisation of PKA and the cAMP signal. We hypothesised that PKA holoenzymes and AKAPs may show spatio-temporal changes in their expression pattern in human myometrium. Western blotting demonstrated a three-fold increase in expression of the regulatory RIIα protein subunit of PKA during pregnancy. RT-PCR and mRNA stability assays confirmed this change was mirrored at the mRNA level. During parturition both transcript and protein are significantly decreased. The functional significance of this was verified by PKA phosphorylation assays. Immunoprecipitation assays demonstrated RIIα complexes with AKAP95 and AKAP79. These findings provide a mechanism to direct the cAMP-quiescence-signal to specific subcellular loci during pregnancy; this effect is then removed during parturition.

Recurrent miscarriage: a possible male factor? A. J. Campbell, P. Bishton, E. H. Gordon and D. S. Irvine. MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, 49 Little France Crescent, Edinburgh EH16 4SB.

Recurrent miscarriage is a distressing condition whose aetiology is often unexplained. Comparatively little attention has been paid to the possibility that abnormalities in the gamete contributed by the male partner could contribute to the origins of early pregnancy failure. The aim of this study was to examine the relationship between human spermatozoal DNA fragmentation and early pregnancy loss. Male partners of women attending a recurrent miscarriage clinic and fertile controls participated. Basic semen analysis was assessed and spermatozoal DNA fragmentation was quantified using the Comet assay and TUNEL assay. There were no differences in basic semen parameters but differences in sperm DNA integrity were detected using both assays. The median (IQR) percentage TUNEL positive sperm in the recurrent miscarriage group was 18.0 (14.1–21.6) and the control group 8.6 (4.7–13.0), (p < 0.001). This data suggests that there may be a relationship between the genetic integrity of the male gamete and the problem of recurrent miscarriage. It emphasises the importance of further research into the role of the male partner in embryo loss.

Sildenafil (Viagra) restores endometrial perfusion in women with unexplained subfertility. N. J. Raine-Fenning, B. K. Campbell, J. S. Clewes, N. R. Kendall and I. R. Johnson. Academic Division of Reproductive Medicine, School of Human Development, University of Nottingham.

Three-dimensional power Doppler angiography was used to quantify the effect of sildenafil on endometrial perfusion (EP) during the menstrual cycle in women with unexplained subfertility (USF). 27 controls and 29 women with USF were randomised to receive either 50mg of sildenafil, administered orally 8-hourly from day 5 to ovulation, or placebo in a double blind, crossover study design. In the control group EP significantly increased (p < 0.001) during the follicular phase peaking 3 days prior to ovulation before decreasing to a nadir day 5 days later (p < 0.001). Thereafter EP gradually increased during the transition from early to mid-luteal phase. Women with USF demonstrated similar trends in EP but with a significantly lower blood flow during the late follicular phase (p < 0.05) and early luteal phase (p < 0.05). Sildenafil was associated with a significant increase in EP (p < 0.05) in both groups. Oestradiol and progesterone assays did not differ between the groups.

Protein Z concentrations in normal pregnancy. Frances Stewart,1 Jane E. Ramsay,1 Hugh Friel,2 Isobel Walker,3 Ian A. Greer,1 Mark D. McColl.21University Department of Obstetrics and Gynaecology, 3Department of Haematology, Glasgow Royal Infirmary. 2Department of Haematology, Crosshouse Hospital, Kilmarnock.

Protein Z (PZ) is a vitamin K dependent glycoprotein that functions as a natural anticoagulant. Data suggests PZ may play a role in fetal loss. In a prospective study we assayed PZ concentrations in all trimesters and 3 months postpartum in 40 women. Mean age was 28.4 (SD5) years and BMI 28.6 (SD6.4) kg/m2. Mean PZ increased from 1.73 mcg/ml (SD0.7) in the first trimester (T1) to 1.96 mcg/ml ((SD0.7), p < 0.001) in T2 and 1.9 mcg/ml (SD0.8) in T3 (p = 0.7 versus T2). Levels fell at 3 months postpartum to 1.3 mcg/ml ((SD0.5), p < 0.001 versus T3; p < 0.001 versus T1). In T3 a positive relationship was noted with T1 waist circumference (r = 0.34, p = 0.03). This was not observed in the non-pregnant period. We conclude that PZ concentrations increase early in pregnancy. This may be a physiological response to control the hypercoagulability of pregnancy. However, failure of this mechanism may predispose to a pro-thrombotic state explaining the association with poor pregnancy outcome.

Continence outcome after fourth degree obstetric injury—a prospective review. Maeve Eogan, Rhona Mahony, Colm O'Herlihy. UCD Department of Obstetrics and Gynaecology, National Maternity Hospital, Holles St, Dublin 2.

Anal sphincter injuries can be classified as third degree (3dt) or fourth degree (4dt) tears. The two grades of injury are often considered together, which limits the advice that can be given postnatally and in subsequent pregnancies.

Data were prospectively accrued on patients attending with a history of a 4dt. Results were compared with those of women who had sustained the anatomically less disruptive laceration, a 3dt.

Demographics and overall continence outcome were similar, but manometry and endoanal ultrasound were more likely to be abnormal after a 4dt.

Within the 4dt group, 6 patients have had a subsequent delivery. Four have delivered vaginally with none experiencing altered faecal continence subsequently.

In conclusion, we have not found fourth degree tear to be associated with significant continence disruption. Furthermore, further vaginal delivery is not always contraindicated when a patient has a history of 4dt.

A prospective community-based cohort study of women undergoing routine cervical screening: A comparison of minichromosome maintenance protein testing, liquid-based cytology and HPV testing. M. E. Flynn,1 S. Robinson,1 A. Sargent,3 L. M. Morris,2 N. Coleman,2 A. Bailey,3 R. A. Laskey,2 H. C. Kitchener.11Academic Unit of Obstetrics and Gynaecology, St Mary's Hospital, Manchester, 2MRC Cancer Cell Unit, Hutchinson/MRC Research Centre, Cambridge. 3Department of Virology, Manchester Royal Infirmary, Manchester.

Liquid-based cytology (LBC) produces a more homogeneous sample than a conventional smear; residual sample can provide material for additional tests. Persistent infection with ‘high-risk’ HPV is an essential precursor for high-grade CIN and cervical carcinoma. Minichromosome maintenance (MCM) proteins are essential for initiating DNA replication and are degraded when the cell exits the cell-cycle. Persistence of MCM proteins is found in pre-malignant and malignant cells. The aim of this study is to evaluate the role of immunocytochemical staining for MCM proteins in an LBC cervical smear, with cytology and HPV testing. All women recruited into the study have a colposcopy/biopsy performed at the time of screening. This serves as the ‘gold-standard’ comparison. Immunocytochemistry is performed using antibodies against MCM 2 & MCM 5. HPV testing is performed using Hybrid Capture (II). 864 women have been recruited. Colposcopy/biopsy was normal in 771 (89%), low grade CIN in 77 (9%) and high grade CIN in 16 (2%). HPV was positive in 114 (13%). The sensitivity, specificity, positive and negative predictive values of cytology and HPV testing to detect high-grade CIN are 94%, 84%, 10% and 99% for cytology; 100%, 88%, 14% and 100% for HPV testing. Preliminary MCM analysis is negative in 84% cases, positive in squamous cells in 12%, positive in endocervical cells in 4%. MCM was positive in 3/3 cases of high grade CIN. In conclusion, both cytology and HPV testing perform well in the detection of high-grade CIN. The role of MCM analysis need to be further elucidated in this cohort.

Angiotensinogen (AGT) gene variants in fetal growth restriction (FGR). C. Tower,1 S. Plummer,1 L. Morgan,1 P. Baker,2 N. Kalsheker.11School of Molecular Medical Sciences, University of Nottingham. 2Maternal and Fetal Health Research Centre, University of Manchester.

Genetic epidemiology suggests a role for genes in the pathogenesis of FGR. A case-control study from Utah found an increased frequency of the AGT 235Met > Thr polymorphism in FGR. We investigated this, and 2 further variants, 174Thr > Met and 11535C > A to define the 4 common AGT haplotypes, in 107 families with FGR and 101 families with normal pregnancies. All subjects were white western Europeans. There was no significant difference in maternal 235Thr allele frequencies in FGR (0.43) compared with controls (0.41; p = 0.35), nor in AGT haplotype frequencies (p > 0.15). Transmission disequilibrium testing of fetal genotype found no distortion of transmission of any haplotype (p > 0.56). This test examines distortion from the expected probability of 0.5 of allele transmission from heterozygous parents to affected offspring. Therefore, in the Nottingham population, AGT haplotype does not affect the risk of FGR. These findings may reflect population differences between Utah and Nottingham.

The variant T allele of the interferon (IFN)-γ+ 874 (A/T) polymorphism increases susceptibility to mild/moderate, but not severe, pre-eclampsia. H. Zghebeh, N. M. Orsi and J. J. Walker. Academic Unit of Paediatrics, Obstetrics and Gynaecology, Leeds General Infirmary, Leeds.

Pre-eclampsia (PET) is an inflammatory disorder affecting 10% of pregnancies. The association between the functional IFN-γ+874 polymorphism and PET was investigated. Pre-eclamptic women >25 weeks' gestation (n = 39; BP > 130) were subdivided into mild/moderate (n = 22; DBP < 110) or severe (n = 17; DBP > 110) and compared to matched control pregnancies (n = 50). Maternal DNA was genotyped by ARMS-PCR. Allele/carrier frequencies in the control and PET groups were similar (0.62 vs. 0.49/0.82 vs. 0.78), while T allele frequency was significantly higher in the mild/moderate subgroup compared to controls (0.70 vs. 0.49; OR 2.48; 95%CI 1.16–5.29; P < 0.05). The mild/moderate subgroup had a significantly higher carrier frequency than the severe (0.95 vs. 0.65; OR 11.45; 95%CI 1.22–107.51; P < 0.05). The T allele increases susceptibility to mild/moderate, but not severe, PET. IFN-γ genotype may influence PET presentation rather than being a causative agent, highlighting the need to identify subpopulations based on disease severity in PET.

17α-hydroxyprogesterone caproate (17P) and human myometrial contractility in vitro. Sexton D. J., O'Reilly M. W., Friel A. M., Morrison J. J. Department of Obstetrics and Gynaecology, Clinical Science Institute, University College Hospital Galway, National University of Ireland Galway.

It has been recently reported that weekly injections of 17P for women who have had a previous spontaneous preterm delivery reduces the risk of preterm delivery. The mechanism of action of 17P is unknown. The aim of this study was to investigate the effects of 17P on isolated human myometrial contractions in vitro, during pregnancy and in the non-pregnant state. Biopsies were obtained at elective cesarean section (n = 11) and at hysterectomy (n = 6). Dissected strips were mounted in tissue baths for isometric recording under physiological conditions. The effects of 17P (10−9M–10−5M) on both human pregnant [spontaneous and oxytocin(0.5nM)-induced contractions] and non-pregnant [spontaneous and phenylephrine (10 μM)-induced contractions] myometrium were investigated There was no significant net relaxant or uterotonic effect exerted by 17P on myometrial contractility. The mean net maximal inhibition (MMI) in pregnant myometrium for spontaneous and oxytocin-induced contractions were 4.91% (p = 0.309) and 2.19% (p = 0.128) respectively. The MMI in non-pregnant myometrium for spontaneous and phenylephrine-induced contractions were 8.773% (p = 0.121) and −7.89% (p = 0.966) respectively. These findings indicate that the reported benefits of 17P for preterm delivery are not achieved by a traditional tocolytic or utero-relaxant effect, and invoke other mechanisms.

Thromboelastographic assessment of whole blood haemostasis in pregnancy—a prospective longitudinal study. Tosin Ajala, Karl Murphy and Raj Rai. Department of Obstetrics and Gynaecology, St Mary's Hospital NHS Trust and Faculty of Medicine, Imperial College London.

Thrombophilic defects may be associated with late pregnancy complications and an inverse relationship between thrombin generation and birthweight has been reported. Thromboelastography is a rapid, reproducible assessment of the kinetics, strength and stability of whole blood haemostasis. We determined the longitudinal changes in whole blood haemostasis amongst 132 consecutive primiparous women with uncomplicated singleton pregnancies. Samples were obtained at four weekly intervals from 22 weeks gestation until delivery. Between 22 and 40 weeks gestation the median coagulation index rose from 3.13 to 4.09 units (p < 0.001); the maximum clot strength from 65.2 mm to 72.4 mm (p < 0.001) and the rate of clot formation from 2.1 to 2.5 mm (p < 0.05). No significant change was demonstrated in the time to initiate coagulation or clot lysis. The increase in coagulation in pregnancy is primarily due to an increase in the coagulation response rather than an impairment of fibrinolysis.

Ultrasound guided delivery of gene therapy to the early gestation fetal sheep brain.1Weisz B., 1David A. L., 1Peebles D. M., 1Perocheau D., 2Themis M., 3Cook T., 2Coutelle C. and 1Rodeck C. H. 1Department of Obstetrics and Gynaecology, Royal Free and University College Medical School, University College London; 2Gene Therapy Research Group, Section of Molecular Genetics and 3Department of Histopathology, Imperial College School of Medicine, London.

Gene therapy of the fetal brain may be useful for the treatment of congenital diseases that may affect the brain from early in fetal development such as the glycogen storage disorder, Tay-Sachs.Ultrasound visualization of the sheep fetal brain was assessed by a preliminary study (n = 95). Adenoviral vectors encoding the β-galactosidase gene (1–2.3 × 1012 p/kg) were injected into the lateral ventricles of early gestation fetal sheep (n = 3, 54–60 days of gestation, term = 145 days) under ultrasound guidance.

Expression of β-galactosidase in the fetal brain, as assessed by X gal staining 2 days after injection, was seen in the choroid plexus, the epithelial lining of the lateral ventricles and the neurocortex. Gene therapy vectors can be delivered to the early gestation fetal brain using a clinically relevant technique and gene transfer to the fetal cortex was achieved.

Sialomucin Complex (MUC4) Immunolocalisation in Normal Endometrium and Endometrial Neoplasia. B. Winter-Roach,1 Subathra Sabaratnam,2 Godfrey Wilson,2 Carolyn Jones,1 Henry Kitchener,1 John Aplin.11Academic Unit of Obstetrics and Gynaecology, University of Manchester, 2Department of Histopathology, Central Manchester Health Care Trust.

The mucin MUC4 (human form of the rat SMC) is up-regulated in some adenocarcinomas and may function as an intra-membrane ligand for the tyrosine kinase receptor erbB2. Atypical hyperplasia is a recognised precursor of endometrial adenocarcinoma. The aim of this study was to map the expression of MUC4 in normal and neoplastic endometrium with 1G8, an antibody raised to MUC4. 85 sections comprising 24 of each proliferative and secretory phase endometrium, 17 atypical hyperplasias and 30 endometrial carcinomas were studied. Vascular endothelial MUC4 expression was consistently strong in the normal endometrium, mirroring the expression of CD34; while in neoplastic endometrium was weak. Glandular MUC4 staining was mostly absent from normal endometrium and atypical hyperplasias in contrast to moderate or strong glandular staining in the well-differentiated (G1) carcinomas. The differences in the straining of atypical hyperplasias and well-differentiated carcinomas illustrate a hitherto un-reported feature of the biology of endometrial neoplasia.

The effect of superoxide anions on human myometrial contractility. G. J. Bugg, I. P. Crocker, T. A. Johnston, P. N. Baker and M. J. Taggart. Maternal and Fetal Health Research Centre, St Mary's Hospital, UK.

Objectives: To test the impact of superoxide anions on contractile function on human myometria. Methods: Hypoxanthine(HX) and xanthine oxidase(XO) was combined to generate superoxide anions in vitro. Spontaneously contracting myometrial strips (5mm, 1.5 mm, 1.5mm) were treated with: (a) HX [1mM] followed by XO [200 uM/ml]; (b) HX [1mM], superoxide dismutase (SOD) [200IU/ml] and then XO [200 uM/ml]; (c) XO [200 uM/ml] alone; (d) hypoxanthine [1mM] followed by SOD [200IU/ml]. Results: HX in combination with XO caused the spontaneous contractile amplitude to increase to 110% (median, interquartile range 101%–131%; p = 0.028) and the contractile integral to increase to 129% (114–162%; p = 0.017) of the contractions in PSS (n = 8). However contractility was not significantly by HX and XO in the presence of SOD (n = 6), by HX alone (n = 14), XO alone (n = 6) or SOD alone (n = 6). Conclusion: Superoxide anions generation increased spontaneous myometrial contractility and may be an important mechanism regulating myometrial contractility during parturition.

FasL is expressed in fallopian tube and induces apoptosis of activated T cells. Illanes S.,a–c Gonzalez P.,b Maisey K.,b Valdes D.,b Imarai M.,baFaculty of Medicine, Universidad de los Andes; bLaboratory of Immunology, Department of Biology, Universidad de Santiago de Chile; cDepartment of Obstetric and Gynaecology, University of Bristol.

The Fallopian tube is subject of immunological and hormonal regulation to avoid the occurrence of unrestrained local inflammatory responses in the presence of an infection. This can be regulated via apoptosis by binding FasL to Fas on the surface of T cells. We examined the expression of FasL in human oviduct in different phases of the menstrual cycle and determined if epithelial cells could induce apoptosis of T cells in vitro. The expression of FasL was assessed by immunofluorescense and Western-Blot. Apoptosis in T cells was quantified by TUNEL and flow cytometry. FasL is expressed in the epithelium and stroma of the oviduct and that fixed epithelial cells induced apoptosis of activated T cells. Our data suggest that the expression of FasL might change in response to the phases of menstrual cycle. These results show that FasL might play a role in the regulation of the genital local immune response.

Antiphospholipid Syndrome(APS), recurrent miscarriage and long-term thrombotic episodes—Is there a link? K. Govardhan Das, R. G. Farquharson, F. Dawood, J. Topping, *S. Quenby. Liverpool Womens Hospital, *Liverpool University.

The objective of this case controlled questionnaire study was to undertake a long-term follow up of thrombotic episodes in women attending a tertiary recurrent miscarriage clinic in the UK. 300 women with primary APS attending the Liverpool Women's Hospital recurrent miscarriage clinic were identified from the clinic database. 140 women returned the questionnaire. These women were matched to controls from the idiopathic section of the clinic database. Validation was performed on the first ten questionnaires. The mean length of follow up was 7.2 years and 7.3 years in the APS and the control groups respectively. Analysis of the results from both groups showed that a past history of thrombosis, recurrent pregnancy loss and positive tests for APS are significant risk factors for long-term serious thrombotic sequelae. Further prospective studies are needed to assess the long-term risk of thrombosis.

The impact of body mass index on the outcome of burch colposuspension for urodynamic stress incontinence. V. Lawton, H. C. Kitchener, A. R. B. Smith. The University of Manchester Academic Unit of Obstetrics and Gynaecology, Human Development and Reproductive Health.

The impact of body weight on cure rates following continence surgery is unknown. This paper examines blinded data at six months follow-up from the MRC CoLPO trial. This trial is a randomised controlled trial comparing open and laparoscopic Burch colposuspension for urodynamic stress incontinence. Women with detrusor overactivity were excluded.The body mass index was recorded for the 291 women recruited to the study. Open or laparoscopic colposuspension was performed on 290 women. Cure was defined as a loss of 1.0g or less on a one-hour ICS pad test.

The effect of BMI on cure was assessed using a univariate logistic regression analysis. Data was available for 225 women were available. The mean BMI was 27.7 kg/m2 (SD 4.8, range 18–45 kg/m2). BMI did not affect the probability of cure (B = 0.037, SE = 0.031, Exp B = 1.037, p = 0.24). Hence overweight women are not less likely to be cured by colposuspension in the short term.

Management and outcome of patients referred with grade 5 smears. N. Gupta, A. Hauke. Department of Obstetrics and Gynaecology, Llandough Hospital, Cardiff, UK.

Objective: The implication of a grade 5 smear result is that possible invasion may be present. Hence should these cases be managed differently to high grade smears (moderate/severe). Setting: Teaching Hospital in Wales. Subjects: 26 patients referred to Llandough Hospital Colposcopy Clinic with grade 5 smears between November 1998 and February 2001.Outcome Measures: Primary management, invasive disease, outcome at 6 months follow up. Results: Primary management included LLETZ (18), two-stage loop excision (2), total abdominal hysterectomy (2), knife cone excision (2) conservative management (1). 5(20%) women had cancer, 2(7.7%) were found to have invasive disease while microinvasive disease was found in 3(11.5%). Conclusion: It is likely that patients with grade 5 smears account for a significantly higher proportion of microinvasive/invasive disease than moderate/severe referrals. These patients should have more urgent referrals, be assessed by experienced colposcopists in order to ensure that invasive disease is not missed and at the same time overintervention is avoided.

Human fetal mesenchymal stem cells for intrauterine cellular therapy. Jerry Chan, Keelin O'Donoghue, Josu de la Fuente, Nigel Kennea, Diana Watt÷ Jennifer Morgan,* Nicholas Fisk. Institute of Reproductive and Developmental Biology, *MRC Clinical Sciences Centre, Imperial College London, London, W12 0NN, UK. ÷University of Brighton and Sussex, Brighton.

To evaluate human fetal mesenchymal stem cells (hfMSC) from first-trimester fetal blood as vehicles for fetal cellular therapy, we studied myogenic differentiation of fMSC in-vitro and in-vivo. Myogenesis was first induced in-vitro by a number of techniques, the highest proportion (>70% desmin and myosin positive giant multinucleated fibres) being achieved with galectin-1. Transplantation into injured mouse muscle showed that hfMSC contributed to muscle regeneration. To evaluate their potential for gene delivery in-utero hfMSC were transduced with either a retrovirus or a lentivirus with 80–98% efficiencies without affecting growth and differentiation potential, indicating retention of stem cell properties. Intra-uterine transplantation in mid-gestation mice to determine stem cell fate showed persistence and engraftment of hfMSC. Thus hfMSC undergo myogenic differentiation under permissive conditions. This work evaluates a novel type of mesenchymal stem cell for allogeneic or autologous fetal therapy of debilitating childhood muscle diseases, such as muscular dystrophy and other mesenchymal diseases.

The role of Gap Junctions in Omental arteries—normal pregnancy and pre-eclampsia. Joanna C. Gillham,1 Louise C. Kenny,1 Jo D. Glazier,2 Philip N. Baker1 and Michael J. Taggart.11Maternal and Fetal Health Research Centre, St Mary's Hospital, University of Manchester, 2Academic Department of Child Health, St Mary's Hospital, University of Manchester.

The endothelium-dependent hyperpolarising factor (EDHF) pathway is thought to be crucial to the enhanced vasodilation of pregnancy, and may be deficient in pre-eclampsia. Our hypothesis is that gap junctions are responsible for EDHF responses. We studied the effect of gap junction inhibition and expression of gap junction connexion proteins. Endothelium-dependent relaxation was assessed by wire myography. After nitric oxide and prostacyclin inhibition, EDHF relaxation in omental arteries was abolished by gap junction inhibition by 18-alpha glycerrhetinic acid. Using RT-PCR, expression of the 3 expected connexins was demonstrated in omental arteries from normal pregnancy. In pre-eclampsia pregnancies, connexins 40, 43 and 37 were identified, but there was a suggestion of decreased expression of 43 and 37.Gap junctions appear to be integral to the EDHF relaxation pathway. Although expression of connexins 40, 43 and 37 was identified normal pregnant and pre-eclampsia vessels, a suggestion of a quantitative difference in connexion expression merits further analysis with real time PCR.

Maternal undernutrition in the rat affects modulation of myogenic reactivity in the uterine arteries in the pregnant offspring. Sukrutha Veerareddy, MRCOG,1,2,* Denise G. Hemmings, PhD,1,* Shaila J. Merchant, MSc,1,* Philip N. Baker, DM2 and Sandra T. Davidge, PhD.11Department of Obstetrics and Gynaecology, Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada, T6G 2S2 and 2Maternal and Fetal Health Research Centre, Manchester, United Kingdom, M13 OJH.

Epidemiological and animal studies have linked adverse nutrition in utero to development of adult onset diseases in the offspring due to fetal programming. The impact of fetal programming on subsequent pregnancy adaptations of female offspring may result in complications of pregnancy, fetal outcome and induce transgenerational effects. Myogenic tone was assessed in the absence and presence of nitric oxide synthase (NOS) inhibitor in resistance-sized uterine arteries of late-gestation pregnant female offspring of control (C, n = 4) and diet restricted (DR, n = 3) rats. A trend to enhanced myogenicity was noted in arteries from pregnant DR compared to those from pregnant C group. Significant modulation of myogenic tone by NOS pathway was observed only in the C group. These results provide evidence that maternal dietary restriction results in altered vascular function of small uterine arteries in pregnant female offspring that may mediate transgenerational effects associated with fetal programming.

A study of the characteristics of the spiral arteries in the placental bed of normal and pre-eclamptic pregnancies. R. A. Duckett,1 W. Dunn,1 P. N. Baker,2 I. R. Johnson.11School of Molecular Medical Sciences, University of Nottingham, 2Maternal and Fetal Health Research Centre, University of Manchester.

The distal spiral arteries in the placental bed undergo physiological change and are crucial to the pathogenesis of pre-eclampsia in which the process is thought to be incomplete. Pressure myography was performed on the distal spiral arteries from the placental beds of normal and pre-eclamptic pregnancies. Normal vessels had a greater lumen diameter, wall thickness and wall to lumen ratio, but a similar distensibility to pre-eclamptic vessels. Possible explanations for this unexpected result are that either there is no significant difference between normal and pre-eclamptic vessels, or that selection bias is introduced at the time of dissection, confirming that physiological change is not an all or nothing phenomenon. Further work is to be performed on whole placental bed biopsies to identify the extent of trophoblast invasion across the whole sample.