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ORAL PRESENTATIONS 1 ORAL PRESENTATIONS SRF PhD Prize Session SRF.1 The novel use of androgens to optimise detection of the fertile window in giant pandas Kirsten Wilsona, Jella Wautersb, Iain Valentinec, Alan McNeillya, Mick Raed, A. Forbes Howiea, Ruth Andrewe, W. Colin Duncana aMRC CRH, University of Edinburgh; bGhent University, Belgium; Pairi Daiza Foundation, Belgium; Leibniz Institute for Zoo and Wildlife Research, Germany; cZoocraft; dEdinburgh Napier University; eBHF CVS, University of Edinburgh Background: Giant pandas are seasonally monoestrus. In Spring, estrogens increase for 1-2 weeks before a peak is reached, followed by 24-48 hours of fertility. Accurately monitoring the hormone profile is therefore vital for prediction and detection of this fertile window to facilitate insemination in a captive breeding programme. Aim: To consider biomarkers for advanced warning of estrus and the accurate timing of insemination. Methods: Urine samples from three females covering 9 breeding cycles, from 40 days before estrus until 10 days after estrus, were assayed for estrone-3-glucuronide (E1G) and progesterone (P4) by Arbor Assays ELISAs, for testosterone (T) and DHEA by validated in-house ELISAs, and for luteinising hormone (LH) by bioassay. Hormone concentrations were corrected against urinary specific gravity (USpG). Results: E1G increased for 11.4 days (range 9-14) before peaking, with the start of the rise detected retrospectively. Prospectively, using current methods, it starts when E1G concentrations become higher than P4 (9.6 days, range 7- 11). Alternatively, E1G becoming higher than T gives 4-days advanced warning of impending estrus (12.7 days, range 9-17). LH was analysed as a marker of peak fertility to time insemination. Only 7/9 cycles showed a surge, ranging -1 to +3 days from peak E1G; thus LH could not reliably predict optimal insemination timing. However, DHEA showed a significant increase only on the day of peak E1G, and not before. Conclusion: Monitoring T alongside P4 can provide 4 days additional warning of upcoming estrus and monitoring DHEA can identify the day of peak E1G without needing to observe a decrease in E1G itself. Hence respectively enlarging the preparative window for captive breeding, facilitating panda management within the ex-situ conservation breeding programme, and unequivocally confirming impending estrus. Therefore, measuring androgens advances and optimises how estrus is monitored in the giant panda. SRF.2 "Horsing around" with gene copy number is associated with equine early pregnancy loss Charlotte A. Shiltona, Brian W. Davisb, D. Claire Wathesa, Terje Raudseppb, Amanda M de Mestrea aRoyal Veterinary College; bTexas A&M University Introduction Copy number variation (CNV) is suggested as necessary to facilitate the rapid growth and differentiation of human placentae, with alterations in CNV associated with human pregnancy loss. The causes of over 60% of equine early pregnancy loss (EPL) are unknown. This study investigated CNV acquisition during normal equine placental development, and identified abnormalities associated with EPL. Methods Extracted DNA from placental tissue of 54 EPLs (14-65 days gestation), 10 age matched clinically normal pregnancies, and 5 term placenta were hybridised to the Axiom Equine 670K SNP Genotyping Array. Probe intensity values (Log2Ratio) were assessed in R (DNAcopy package) to generate CNV calls. Calls were filtered to include only autosomal CNVs of Log2Ratio ±0.2, p<0.05, and over 1 kb in length. Calls within 5 kb were merged. DNA quality was assessed to minimize false positives as a result of lower quality DNA. Results The average number (±SD) of autosomal CNVs increased over time across pre-implantation (25.5±5.3 CNVs), post-implantation (32.3±6.9) and term (34.4±5.1) placenta. EPLs prior to implantation (<40 days) had significantly increased CNVs compared to age matched controls (246.9±439.2 and 25.5±5.3 respectively, p=0.02). This increase was across autosomes with 14.8% of EPLs (n=8/54) having an extreme number of CNVs (range 362-1323) compared with controls (n=10, range 19-41). Conclusion CNV studies in human placental development and pregnancy loss are limited by sample size, and placental tissue differences, making it difficult to draw conclusions. This first study of CNV in equine placentae found that similarly to the limited human studies, equine placentae require increasing CNVs to facilitate rapid expansion. Equine EPLs were associated with extremely high CNVs, a phenomenon not previously described in human pregnancy loss (associated with lower number of CNVs). Further investigation therefore is warranted to establish whether this also occurs in human placentae associated with miscarriage. 2 ORAL PRESENTATIONS SRF.3 The effect of repeated doses of doxorubicin on the pre-pubertal mouse testis Kathleen Duffin, Federica Lopes, Rod Mitchell, Norah Spears University of Edinburgh Background: Increasing childhood cancer survival rates mean more adults living with the consequences of treatment, including infertility (1). For pre-pubertal boys, there are no fertility preservation options available, increasing the importance of understanding effects of chemotherapy on the immature testis. Experimental models usually test the effect of single exposure to a drug; however, clinically, chemotherapy drugs are given at repeated intervals. This work aims to assess whether multiple doses of doxorubicin cause more damage to the germ cells, specifically spermatogonial stem cells (SSCs), than the same cumulative dose given in a single exposure. Methods: Testes fragments from pnd5 mice (n=8) were cultured on polycarbonate membrane floating on medium (α- MEM,10%KSR) at 34°C; 5% CO2 (2,3). Tissue was randomly allocated to receive: control; single dose of doxorubicin (0.1µg/ml) on D2 or D5; or two half doses (0.05µg/ml), on both D2 and D5. On D8, BrdU was added to medium. Immunohistochemistry identified germ cells/MVH+, SSCs/PLZF+, and proliferating SSCs/BRDU+PLZF+. Results: All treatments of doxorubicin caused significant loss of SSCs, with no difference between treatment groups. Only the single dose on D2 caused a significant reduction in density of proliferating SSCs. Doxorubicin caused significant germ cell loss when given in a single dose on D2 or in two half doses on D2 and D5; the single dose on D5 did not cause a significant loss of germ cells. Discussion: Doxorubicin causes significant loss of SSCs, with no differences between regimens, suggesting that cumulative dose could be a key factor in determining doxorubicin-induced damage in the pre-pubertal mouse testis. Overall, however, tissue treated on D2 consistently showed more damage than tissue treated on D5. Ongoing work is investigating whether this is due to time in culture post-exposure, and/or to testicular developmental stage at time of exposure. 1. Chow EJ, Stratton KL, Leisenring WM, Oeffinger KC, Sklar CA, Donaldson SS, et al. Pregnancy after chemotherapy in male and female survivors of childhood cancer treated between 1970 and 1999: A report from the Childhood Cancer Survivor Study cohort. Lancet Oncol. 2016 May 1;17(5):567- 76. 2. Smart E, Lopes F, Rice S, Nagy B, Anderson RA, Mitchell RT, et al. Chemotherapy drugs cyclophosphamide, cisplatin and doxorubicin induce germ cell loss in an in vitro model of the prepubertal testis. Sci Rep. 2018;8(1):1773. 3. Sato T, Katagiri K, Gohbara A, Inoue K, Ogonuki N, Ogura A, et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature. 2011;471(7339):504-7. SRF.4 MiR-371a disrupts the stroma-epithelium communication in human endometrium Dimitra Makri, Marcos Castellanos-Uribe, Sean May, Walid Maalouf University of Nottingham Purpose/background: Members of the miR-290/miR-371 cluster are of the earliest de novo synthesised by embryos and have important actions in mammalian reproduction. Stroma-epithelium communication at the peri-implantation window is necessary to prepare the endometrium to accept an invading embryo. While growth factors, hormones, and cytokines are well-known mediators of this crosstalk, the roles of miRNAs at the embryo-maternal interactome are less clear. Here we investigated the effects of miR-371a in the transcriptome of endometrial stromal cells with specific focus on processes related to the uterine cell communication. Methods: The miR-371a-3p mimic was transfected in human endometrial stromal cells using lipid-based transfection reagents at a 30nM concentration for 72 h. The cells were collected and RNA was extracted and sent for microarray analysis with the Clariom™ S Human Arrays. The Partek® software and online bioinformatics tools were used to identify the top differentially expressed genes, affected processes, and altered pathways. Results: A total of 4.760 genes were altered by miR-371a, which disrupted cell communication, immune response, damage response, binding and enzyme activities and pathways related to signalling -- MAPK, chemokine, p53, Wnt, TNF, and oestrogen. Specific stroma-epithelium communication genes were significantly dysregulated, with downregulation of 7 fibroblast growth factors (FGF-1, -5, -7, -9, -17, -20), the HAND2 stromal factor, progesterone receptor PGR, and insulin growth factor IGF-1. Conclusions: MiR-371a at the embryo-maternal interface causes major changes in the endometrial transcriptome with immediate effects in biological, cellular, and molecular processes which disrupt the stroma-epithelium communication. This disruption could impair the process of implantation,
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