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Fertility 2020 Full Abstract Book Fertility2020 Reproduction in a changing world 9 – 11 January 2020 The AssociationACE of Clinical Embryologists EICC Edinburgh www.fertilityconference.org Abstract Book #Fertility2020 Fertility2020 Gold Sponsors Associated Fertility Societies ORAL PRESENTATIONS SRF POST DOCTORAL PRIZE SESSION SRF.1 Tgfβ/smad signaling regulates granulosa cell proliferation and tzp generation during early follicular development Granados Aparici Sofia; Yang Qin; Clarke Hugh J. Research Institute - McGill University Health Centre; McGill University; Montreal, Quebec, Canada During initiation of follicular development, the somatic cells surrounding the oocyte, termed granulosa cells (GCs), start to proliferate and cuboidalise. Shortly after, an extracellular layer called the zona pellucida starts to form and physically separates the oocyte from the GCs. Since GC-oocyte contact-dependent communication is essential for oocyte development, GCs generate transzonal projections (TZPs), filopodia-like structures that penetrate the zona pellucida and establish contact with the oocyte plasma membrane. Since the TGFβ superfamily of ligands have a well-known role in granulosa function in growing follicles, we sought to investigate whether the canonical mediators, the SMAD3/4, regulate the rate of initial GC proliferation as well as the generation of TZPs in preantral follicles. Quantitative RT-PCR and immunofluorescent histological sections of mouse ovaries showed expression and localisation of Smad3/4 in GCs of preantral follicles from non-growing stage onwards. The tamoxifen-inducible Cre-ER/floxed Smad4+/+ knockout (KO) system was used in neonatal ovaries and granulosa cell-oocyte complexes (GOCs) in culture to delete the Smad4 gene in non-growing and growing follicles. Whole ovaries and GOCs were exposed to tamoxifen (1ug/ml) for 1 and 2 days, respectively, followed by additional days in tamoxifen-free conditions. Wholemount staining of isolated follicles and GOCs was performed and confocal images were taken to quantitatively analyse the GC number, oocyte diameter and TZP number using Image J software. Single-layered follicles isolated from Cre+ ovaries after culture showed a significant decrease in GC number per oocyte diameter when compared to Cre- controls. Cre+ GOCs showed a significant decrease in the number of TZPs whereas oocyte diameter remained unchanged. Our results suggest a new role for the canonical SMAD signalling on the molecular mechanisms regulating early GC proliferation and germ line-somatic communication in preantral follicles which may help identify novel markers of follicle/oocyte quality and provide insight into the causes of infertility. SRF.2 Differential follicle stimulating hormone glycosylation modulates follicle growth and survival rates Johnson Gillian P1; Bousfield George R2; Hardy Kate3; Jonas Kim C1 1King's College London; 2Wichita State University, Kansas; 3Imperial College London Ovarian ageing is a naturally occurring physiological process, marked by dynamic changes in ovarian function and hormone secretion. Several pathologies are associated with ovarian senescence, including; osteoporosis, diabetes, cardiovascular disease and impaired cognitive function, therefore, understanding the physiological processes regulating this is imperative for identifying novel treatments. A key endocrine regulator of ovarian function is the heterodimer glycoprotein hormone, follicle stimulating hormone (FSH). FSH is secreted as two glycosylation variants; partially glycosylated FSH(FSH21) and fully glycosylated FSH (FSH24). These variants have different bioactivities, with FSH21 more biologically active than FSH24. Interestingly, the ratio of FSH21:FSH24 changes with age, with FSH21 predominant in women of reproductive prime, and FSH24 predominant in menopausal women. Yet, if these FSH glycosylation variants differentially modulate follicle growth and survival remains unknown. This study therefore aimed to determine the effects of FSH21 and FSH24 on follicle growth and survival. To do this, mouse ovarian follicles were isolated from 3- 4wk-old-C57/BL6 mice and treated +/- 10ng/ml, FSH21(n=74), FSH24(n=66), a ratio of FSH21:FSH24 at 80:20 (to mimic reproductive prime; n=68) or FSH21:FSH24 at 20:80 (to mimic late peri-menopause; n=66). Follicles were cultured for up to 96hrs and imaged daily to evaluate follicle morphology. Follicle growth was markedly increased at all time points, when cultured in the presence of FSH21 or 80:20 FSH21:FSH24, in comparison to control, FSH24 alone and 20:80 FSH21:FSH24 conditions. Follicles treated with FSH24 or FSH21:FSH24 at 20:80 tended to undergo basement membrane rupture and oocyte extrusion. Moreover, survival rates were significantly decreased in follicles treated with FSH24 or FSH21:FSH24 at 20:80. These data suggest that the nature of FSH glycosylation modulates the follicular cellular environment to regulate follicle growth and survival. These findings have important implications for IVF ovarian hyperstimulation treatment regimens. Moreover, the ratio of FSH21:FSH24 may be an important novel biomarker of ovarian ageing. 1 SRF.3 Exploration of chemerin system in human granulosa cells: involvement in polycystic ovarian syndrome Estienne Anthony; Mellouk Namya; Bongrani Alice; Ramé Christelle; Guérif Fabrice; Froment Pascal; Dupont Joelle INRA Chemerin is an adipokine involved in several metabolic processes. It binds three G protein-coupled receptors: CMKLR1, GPR1, and CCRL2. Recently, a role for the chemerin system in ovarian follicle function and steroidogenesis has been reported in mammals. In both human primary granulosa (hCGs) and human ovarian granulosa-like tumour cell line (KGN), chemerin inhibited IGF-1-induced steroids production. Thus, we investigated chemerin system in hCGs from PCOS patients and KGN and modulation of its signaling activities using a CMKLR1 nanobody (C4910). Our study showed that chemerin mRNA was highly expressed in hCGs of obese, PCOS-O and ECHO-O patients as compared to control, PCOS and ECHO patients and we observed opposite profile for CMKLR1. The mRNA expression of CCRL2 was lower in PCOS, obese and ECHO-O patients as compared to control, ECHO and PCOS-O. In parallel, cultured KGN cells and hCGs were stimulated or not with human recombinant chemerin and/or with C4910. We found that in KGN cells CMKLR1 and CCRL2 receptors formed a heterodimer after 5 minutes of stimulation with chemerin and gradually increased until 60 minutes. We also showed that chemerin rapidly activates MAPK ERK 1/2, P38 and Akt phosphorylation and more slowly AMPK and β-arrestin phosphorylation in KGN cells. Experiments on KGN cell co-incubated with chemerin and the C4910 showed a lower phosphorylation of P38 after 5 minutes than in cells only incubated with chemerin. Experiments on KGN and hGCs co-incubated with chemerin and C4910 showed a reduced progesterone production with a reversal effect of co-incubation with C4910. Our results indicated that chemerin system is expressed in granulosa cells and a deficit of CMKLR1 seems to protect against follicular development disruptions in PCOS patients. We also described the chemerin signaling involved in inflammation and steroidogenesis that was partially blocked by a potential therapeutic nanobody targeting CMKLR1. SRF.4 Aberrant Igf2-H19 expression in the placental endocrine zone increases the susceptibility of the mother to poor metabolic health Lopez-Tello Jorge; Yong Hannah EJ; Christoforou Efthimia R; Napso Tina; Sandovici Ionel; Constancia Miguel; Sferruzzi- Perri Amanda N Centre for Trophoblast Research During pregnancy, the mother must adapt metabolically to support offspring growth. Failures in maternal metabolic adaptation can result in gestational diabetes, which is a risk factor for type-2 diabetes in later life. The placenta secretes hormones with metabolic effects, although the precise role of its endocrine function in determining maternal health is largely unknown. Previous work has shown that the imprinted Igf2-H19 locus is involved in controlling placental endocrine function and conceptus development in mice. This study used conditional mis-expression of the Igf2-H19 locus, through deletion of the imprinting control region ICR1, to induce placental endocrine malfunction and study its consequences for maternal metabolism during and after pregnancy. Mice were crossed to produce entire litters with reduced levels of the H19 gene and activation of the normally silent maternal Igf2 gene in the placental endocrine zone (H19DMRflox/TpbpaCre; Jz-ICR1Δ). On day 16 of pregnancy, dams were terminally anaesthetised for blood collection and tissues collected for molecular analysis. Other dams delivered and, at eight weeks post-partum, they underwent a glucose tolerance test followed by tissue collection one week later. Data were compared to dams with unaltered placental Igf2-H19 locus expression. Jz-ICR1Δ dams had heavier kidney, heart and liver weights compared to pregnant controls. They also displayed higher circulating levels of glucose, insulin, leptin, progesterone, estradiol and corticosterone. Proteins involved in glucose handling (e.g. PI3K-p110α, total AKT and p70S6K) were downregulated in the liver of Jz-ICR1Δ dams. The placental endocrine zone was increased by Jz-ICR1Δ, but maternal plasma IGF2 and fetal growth were unchanged. Dams that carried Jz-ICR1Δ placentas had lower levels of adiposity and were glucose intolerant at 8-9 weeks after delivery. In conclusion, genetically-induced expansion of the placental endocrine zone alters
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