Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCG beta-subunit (LHB), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates LHB promoter activity. Here, studies of the LHB proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the LHB promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp LHB promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the LHB promoter but also requires the upstream segment of the LHB promoter between -119 bp and -335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the LHB promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate LHB transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.

Equine chorionic girdle trophoblast cells play important endocrine and immune functions critical in supporting pregnancy. Very little is known about the genes and pathways that regulate chorionic girdle trophoblast development. Our aim was to identify genes and signalling pathways active in vivo in equine chorionic girdle trophoblast within a critical 7-days window. We exploited the late implantation of the equine conceptus to obtain trophoblast tissue. An Agilent equine 44K microarray was performed using RNA extracted from chorionic girdle and chorion (control) from equine pregnancy days 27, 30, 31 and 34 (n = 5), corresponding to the initiation of chorionic girdle trophoblast proliferation, differentiation and migration. Data were analysed using R packages limma and maSigPro, Ingenuity Pathway Analysis and DAVID and verified using qRT-PCR, promoter analysis, western blotting and migration assays. Microarray analysis showed gene expression (absolute log FC >2, FDR-adjusted P < 0.05) was rapidly and specifically induced in the chorionic girdle between days 27 and 34 (compared to day 27, day 30 = 116, day 31 = 317, day 34 = 781 genes). Pathway analysis identified 35 pathways modulated during chorionic girdle development (e.g. FGF, integrin, Rho GTPases, MAPK) including pathways that have limited description in mammalian trophoblast (e.g. IL-9, CD40 and CD28 signalling). Rho A and ERK/MAPK activity was confirmed as was a role for transcription factor ELF5 in regulation of the CGB promoter. The purity and accessibility of chorionic girdle trophoblast proved to be a powerful resource to identify candidate genes and pathways involved in early equine placental development.

Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family that are secreted by trophoblast cells. PSGs may modulate immune, angiogenic and platelet responses during pregnancy. Until now, PSGs are only found in species that have a highly invasive (hemochorial) placentation including humans, mice and rats. Surprisingly, analyzing the CEACAM gene family of the horse, which has a non-invasive epitheliochorial placenta, with the exception of the transient endometrial cups, we identified equine CEACAM family members that seem to be related to PSGs of rodents and primates. We identified seven genes that encode secreted PSG-like CEACAMs Phylogenetic analyses indicate that they evolved independently from an equine CEACAM1-like ancestor rather than from a common PSG-like ancestor with rodents and primates. Significantly, expression of PSG-like genes (CEACAM44, CEACAM48, CEACAM49 and CEACAM55) was found in non-invasive as well as invasive trophoblast cells such as purified chorionic girdle cells and endometrial cup cells. Chorionic girdle cells are highly invasive trophoblast cells that invade the endometrium of the mare where they form endometrial cups and are in close contact with maternal immune cells. Therefore, the microenvironment of invasive equine trophoblast cells has striking similarities to the microenvironment of trophoblast cells in hemochorial placentas, suggesting that equine PSG-like CEACAMs and rodent and primate PSGs have undergone convergent evolution. This is supported by our finding that equine PSG-like CEACAM49 exhibits similar activity to certain rodent and human PSGs in a functional assay of platelet-fibrinogen binding. Our results have implications for understanding the evolution of PSGs and their functions in maternal-fetal interactions.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Early pregnancy loss occurs in 6–10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14–65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in AmnioChrome™ Plus. Genomic DNA of a suitable quality for molecular assays was also isolated from 29/30 of these cases. This method will enable future investigations determining pathologies causing EPL.

TGFβ superfamily proteins, acting via SMAD (Sma- and Mad-related protein)2/3 pathways, regulate placental function; however, the role of SMAD1/5/8 pathway in the placenta is unknown. This study investigated the functional role of bone morphogenetic protein (BMP)4 signaling through SMAD1/5 in terminal differentiation of primary chorionic gonadotropin (CG)-secreting trophoblast. Primary equine trophoblast cells or placental tissues were isolated from day 27–34 equine conceptuses. Detected by microarray, RT-PCR, and quantitative RT-PCR, equine chorionic girdle trophoblast showed increased gene expression of receptors that bind BMP4. BMP4 mRNA expression was 20- to 60-fold higher in placental tissues adjacent to the chorionic girdle compared with chorionic girdle itself, suggesting BMP4 acts primarily in a paracrine manner on the chorionic girdle. Stimulation of chorionic girdle-trophoblast cells with BMP4 resulted in a dose-dependent and developmental stage-dependent increase in total number and proportion of terminally differentiated binucleate cells. Furthermore, BMP4 treatment induced non-CG-secreting day 31 chorionic girdle trophoblast cells to secrete CG, confirming a specific functional response to BMP4 stimulation. Inhibition of SMAD2/3 signaling combined with BMP4 treatment further enhanced differentiation of trophoblast cells. Phospho-SMAD1/5, but not phospho-SMAD2, expression as determined by Western blotting was tightly regulated during chorionic girdle trophoblast differentiation in vivo, with peak expression of phospho-SMAD1/5 in vivo noted at day 31 corresponding to maximal differentiation response of trophoblast in vitro. Collectively, these experiments demonstrate the involvement of BMP4-dependent pathways in the regulation of equine trophoblast differentiation in vivo and primary trophoblast differentiation in vitro via activation of SMAD1/5 pathway, a previously unreported mechanism of TGFβ signaling in the mammalian placenta.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

s 219 © 2013 Th e British Fertility Society A2.3: Mesoporous silica nanoparticle (MSNP)mediated delivery to mammalian sperm and oocytes: localisation, viability and loading studies Natalia Barkalina, Junaid Kashir, Caroline Day, Celine Jones, Helen Townley & Kevin Coward Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women ’ s Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK A2.4: CatSper and stored Ca2 control functionally different ‘ behaviours ’ in human sperm Stephen Publicover, Wardah Alasmari, Sarah Costello, Joao Correia, Senga Oxenham, Leonor Fernandes & Jackson Kirkman-Brown School of Biosciences and Birmingham Women’s Fertility Centre, University of Birmingham, Edgbaston, Birmingham,UK A2.5: Modulation of human sperm motility Joao Correia & Stephen Publicover School of Biosciences, University of Birmingham, Edgbaston, Birmingham,UK A3.1: Development, implementation and clinical outcomes of a multiple birth minimisation strategy Jenny Spencer, Nicolas Vulliemoz, Tim Child, Enda McVeigh & Karen Turner Oxford Fertility, Institute of Reproductive Sciences, Oxford, UK A3.2: Female genital mutilation: are we meeting the standards of antenatal management Munawar Hussain, Charlotte Jones & Jane Booth St.Michael’s Hospital & Bristol Centre for Reproductive Medicine Bristol, Bristol University Medical School, Bristol, UK A3.3: Use of neonatal care (NICU) services after ART: a comparison of singleton and multiple births Munawar Hussain, Kate Lightly, Erik Lenguerrand & Peter Wardle Bristol Centre for Reproductive Medicine, St.Michael’s Hospital Bristol, Southmead Hospital Bristol, Bristol University, Bristol, UK A3.4: Should we be freezing embryos? Mohar Goswami, Erica Haimes & Alison Murdoch Newcastle Fertility Centre At Life, Policy, Ethics and Life Sciences Research Centre, Newcastle University, Newcastle upon Tyne, UK A3.5: The role of the ‘ specialist ’ Fertility Nurse in the establishment and maintenance of a service treating patients with known infectious disease Sally Wood, Grace Marsden, Bijal Patel, Steve Troup, Charles Kingsland & Andrew Drakeley Hewitt Fertility Centre, Liverpool Women ’ s NHS Foundation Trust, Liverpool, UK A3.6: Supporting emotional well-being – Impacts on Assisted Reproductive Technology (ART) nursing practice Judith Applegarth 1 , Trudy Dwyer 2 & Lorna Moxham 3 1 Monash IVF, Rockhampton, Queensland, Australia, 2 Central Queensland University School of Nursing & Midwifery and Institute of Health & Social Science Research, Rockhampton, Queensland, Australia, and 3 University of Woolongong, School of Nursing, Midwifery and Indigenous Health, Wollongong, NSW, Australia A4.1: Heparin treatment for unexplained recurrent implantation failure: empirical use or evidence based? Neelam Potdar, Jane Blower & Tarek Gelbaya Leicester Fertility Centre, University Hospitals of Leicester, Leicester, UK A4.2: Transforming growth factorb family regulation of inhibitor of differentiation proteins in the peritoneal mesothelium: a potential explanation for the aetiology of endometriosis Vicky Jane Young, Jeremy Brown, Christopher Harlow, Alan McNeilly, W. Colin Duncan & Andrew Horne MRC Centre for Reproductive Health, The University of Edinburgh, The Queen ’ s Medical Research Institute, Edinburgh, UK A4.3: Follicular fl uid cytokine dynamics in natural cycles N. Ellissa Baskind, Vinay Sharma, Nicolas Orsi & Nigel Simpson Leeds Centre for Reproductive Medicine, Leeds Teaching Hospitals NHS Trust; University of Leeds, Leeds, UK A4.4 : The ex vivo effect of uterine fi broids on prostaglandins in menorrhagic women Debbie Fischer 1 , Joanne Garvin 2 , Diane Farrar 3 , Peter O’Donovan 3 , David Woodward 2 & Kay Marshall 2 1 School of Pharmacy and Pharmaceutical Sciences , University of Manchester, Manchester, UK, 2 University of Bradford, Bradford, UK and 3 Bradford Royal Infi rmary, Bradford, UK A4.5: Essure “ for management of hydrosalpinges prior to IVF – A systematic review and pooled analysis Puneet Arora 1 & David Cahill 2 1 Department of Reproductive Medicine, St Mary’s Hospital, Manchester, UK and 2 Department of Obstetrics and Gynaecology, St Michael’s Hospital, Bristol, UK A5.1: Anti-Mullerian hormone, polycystic ovarian morphology and polycystic ovary syndrome: A prospective cohort study Roy Homburg, Arpita Ray, Priya Bhide, Amit Shah, Anil Gudi, Peter Timms & Kate Grayson Homerton Fertility Centre, Homerton University Hospital, London, UK

In target tissues, cortisol is metabolised by two 11β-hydroxysteroid dehydrogenase (11βHSD) isoenzymes, namely 11βHSD1 and 11βHSD2, both of which are co-expressed in the boar testis and reproductive tract. The present study has assessed whether cortisol-cortisone metabolism in boar testis and caput epididymidis can be regulated via the gonadotrophin-cAMP signalling pathway. 11βHSD activities were measured by using a radiometric conversion assay in static tissue culture. In both testis and caput epididymidis, the net reduction of cortisone but not the net oxidation of cortisol, was significantly decreased by luteinising hormone (by 53 ± 20% and 45 ± 9%, respectively, P < 0.05), forskolin (by 60 ± 7% and 57 ± 9%, respectively, P < 0.01) and 8-bromo-cAMP (by 54 ± 4% and 64 ± 1%, respectively, P < 0.01). This suppression of 11-ketosteroid reductase activity in the boar testis by forskolin could be attenuated by the protein kinase A (PKA) inhibitor, H89. Hence, within the boar testis and the caput epididymidis, the local actions of glucocorticoids are modulated by gonadotrophin-cAMP-PKA signalling via their selective effects on the reductase activity of 11βHSD.

Tree projections provide a mathematical framework that encompasses all the various (purely) structural decomposition methods that have been proposed in the literature to single out classes of nearly-acyclic (hyper)graphs, such as the tree decomposition method, which is the most powerful decomposition method on graphs, and the (generalized) hypertree decomposition method, which is its natural counterpart on arbitrary hypergraphs.The paper analyzes this framework, by focusing in particular on “minimal” tree projections, that is, on tree projections without useless redundancies. First, it is shown that minimal tree projections enjoy a number of properties that are usually required for normal form decompositions in various structural decomposition methods. In particular, they enjoy the same kind of connection properties as (minimal) tree decompositions of graphs, with the result being tight in the light of the negative answer that is provided to the open question about whether they enjoy a slightly stronger notion of connection property, defined to speed-up the computation of hypertree decompositions. Second, it is shown that tree projections admit a natural game-theoretic characterization in terms of the Robber and Captain game. In this game, as for the Robber and Cops game characterizing tree decompositions, the existence of winning strategies implies the existence of monotone ones. As a special case, the Robber and Captain game can be used to characterize the generalized hypertree decomposition method, where such a game-theoretic characterization was missing and asked for. Besides their theoretical interest, these results have immediate algorithmic applications both for the general setting and for structural decomposition methods that can be recast in terms of tree projections.