P–181 Morphine regulates BMP4 growth factor and is involved in in-vitro early embryo development and PGCs formation

Abstract Study question To elucidate if morphine can alter embryo development. Summary answer Chronic morphine treatment regulates BMP4 growth factor, in terms of gene expression and H3K27me3 enrichment and promotes in-vitro blastocysts development and PGC formation. What is known already BMP4 is a member of the bone morphogenetic protein family, which acts mainly through SMAD dependent pathway, to play an important role in early embryo development. Indeed, BMP4 enhances pluripotency in mouse embryonic stem cells (mESCs) and, specifically, is involved in blastocysts formation and primordial germ cells (PGCs) generation. Although, external morphine influence has been previously reported on the early embryo development, focus on implantation and uterus function, there is a big concern in understanding how environmental factors can cause stable epigenetic changes, which could be maintained during development and lead to health problems. Study design, size, duration First, OCT4-reported mESCs were chronically treated with morphine during 24h, 10–5mM. After morphine removal, mESCs were collected for RNA-seq and H3K27me3 ChIP-seq study. To elucidate the role of morphine in early embryo development, two cell- embryos stage were chronically treated with morphine for 24h and in-vitro cultured up to the blastocyst stage in the absence of morphine. Furthermore, after morphine treatment mESCs were differentiated to PGCs, to elucidate the role of morphine in PGC differentiation. Participants/materials, setting, methods Transcriptomic analyses and H3K27me3 genome wide distribution were carried out by RNA-Sequencing and Chip-Sequencing respectively. Validations were performed by RNA-RT-qPCR and Chip-RT-qPCR. Main results and the role of chance Dynamic transcriptional analyses identified a total of 932 differentially expressed genes (DEGs) after morphine treatment on mESCs, providing strong evidence of a transcriptional epigenetic effect induced by morphine. High-throughput screening approaches showed up Bmp4 as one of the main morphine targets on mESCs. Morphine caused an up-regulation of Bmp4 gene expression together with a decrease of H3K27me3 enrichment at promoter level. However, no significant differences were observed on gene expression and H3K27me3 enrichment on BMP4 signaling pathway components (such as Smad1, Smad4, Smad5, Smad7, Prdm1 and Prmd14) after morphine treatment. On the other hand, the Bmp4 gene expression was also up-regulated in in-vitro morphine treated blastocyst and in-vitro morphine treated PGCs. These results were consistent with the increase in blastocyst rate and PGC transformation rate observed after morphine chronic treatment. Limitations, reasons for caution To perform the in-vitro analysis. Further studies are needed to describe the whole signaling pathways underlying BMP4 epigenetic regulation after morphine treatment. Wider implications of the findings: Our findings confirmed that mESCs and two-cell embryos are able to memorize morphine exposure and promote both blastocyst development and PGCs formation through potentially BMP4 epigenetic regulation. These results provide insights understanding how environmental factors can cause epigenetic changes during the embryo development, leading to alterations and producing health problems/diseases Trial registration number Not applicable </jats:sec

O-148 Sperm Aminopeptidase N as a predictive biomarker of blastocyst development and embryo viability

Abstract Study question To evaluate human sperm APN as a prognostic factor for determining high-quality embryos. Summary answer The human sperm APN has the potential to become new molecular prognostic biomarker for having high-quality and viable embryos. What is known already Prognosis and diagnosis of male fertility is one of the major concerns in reproductive medicine. Approximately 30%-40% of men with otherwise normal fertility parameters are still unable to achieve pregnancy. The predictive clinical value of a semen analysis to identify fertile or infertile males is limited; therefore, new sperm diagnostic or prognostic methodologies are urgently required. Sperm Aminopeptidase N (APN) may be a relevant molecular marker due to its high concentration in sperm cells and its role in sperm physiology, such as motility, acrosome reaction, and embryo development. Study design, size, duration A prospective study that involves a total of 81 couples and 611 embryos who underwent oocyte-donation cycles at the Clínica IVI Bilbao (Spain) between September 2014 and July 2015. Participants/materials, setting, methods This study was set in an assisted reproduction unit and in an academic research laboratory. All semen samples were examined and classified following WHO guidelines. Spermatozoa were isolated from semen on discontinuous colloidal silica gradient (45%-90%) technique. Embryo quality and development were determined according to the Spanish Association of Reproduction Biology Studies (ASEBIR) criteria. Flow cytometry analyses of quantitative and semi-quantitative sperm human APN levels. Main results and the role of chance The obtained results proved that the most evolved and viable blastocysts were associated with low sperm APN levels. Expanding, expanded, hatching/hatched and viable blastocysts come from semen samples which showed lower APN levels than early blastocysts, blocked and non viable blastocyst. The cumulative probability of having more evolved blastyocysts increased 1.38-fold at day 5 and 1.98-fold at day 6 of embryo development as well as the likelihood of having viable embryo increased 1.48-fold when semen samples with low APN levels are used during the ICSI technique. Limitations, reasons for caution Data obtained from a single Fertility Clinic. A multi-centrum study will be required. Wider implications of the findings The human sperm APN has the potential to become new molecular prognostic biomarker for having high-quality embryos that could help to diagnose male infertility, especially when seminal parameters are close to the threshold values. Trial registration number Not applicable </jats:sec

P-181 Morphine regulates BMP4 growth factor and is involved in in-vitro early embryo development and PGCs formation

Abstract Study question To elucidate if morphine can alter embryo development. Summary answer Chronic morphine treatment regulates BMP4 growth factor, in terms of gene expression and H3K27me3 enrichment and promotes in-vitro blastocysts development and PGC formation. What is known already BMP4 is a member of the bone morphogenetic protein family, which acts mainly through SMAD dependent pathway, to play an important role in early embryo development. Indeed, BMP4 enhances pluripotency in mouse embryonic stem cells (mESCs) and, specifically, is involved in blastocysts formation and primordial germ cells (PGCs) generation. Although, external morphine influence has been previously reported on the early embryo development, focus on implantation and uterus function, there is a big concern in understanding how environmental factors can cause stable epigenetic changes, which could be maintained during development and lead to health problems. Study design, size, duration First, OCT4-reported mESCs were chronically treated with morphine during 24h, 10-5mM. After morphine removal, mESCs were collected for RNA-seq and H3K27me3 ChIP-seq study. To elucidate the role of morphine in early embryo development, two cell- embryos stage were chronically treated with morphine for 24h and in-vitro cultured up to the blastocyst stage in the absence of morphine. Furthermore, after morphine treatment mESCs were differentiated to PGCs, to elucidate the role of morphine in PGC differentiation. Participants/materials, setting, methods Transcriptomic analyses and H3K27me3 genome wide distribution were carried out by RNA-Sequencing and Chip-Sequencing respectively. Validations were performed by RNA-RT-qPCR and Chip-RT-qPCR. Main results and the role of chance Dynamic transcriptional analyses identified a total of 932 differentially expressed genes (DEGs) after morphine treatment on mESCs, providing strong evidence of a transcriptional epigenetic effect induced by morphine. High-throughput screening approaches showed up Bmp4 as one of the main morphine targets on mESCs. Morphine caused an up-regulation of Bmp4 gene expression together with a decrease of H3K27me3 enrichment at promoter level. However, no significant differences were observed on gene expression and H3K27me3 enrichment on BMP4 signaling pathway components (such as Smad1, Smad4, Smad5, Smad7, Prdm1 and Prmd14) after morphine treatment. On the other hand, the Bmp4 gene expression was also up-regulated in in-vitro morphine treated blastocyst and in-vitro morphine treated PGCs. These results were consistent with the increase in blastocyst rate and PGC transformation rate observed after morphine chronic treatment. Limitations, reasons for caution To perform the in-vitro analysis. Further studies are needed to describe the whole signaling pathways underlying BMP4 epigenetic regulation after morphine treatment. Wider implications of the findings Our findings confirmed that mESCs and two-cell embryos are able to memorize morphine exposure and promote both blastocyst development and PGCs formation through potentially BMP4 epigenetic regulation. These results provide insights understanding how environmental factors can cause epigenetic changes during the embryo development, leading to alterations and producing health problems/diseases. Trial registration number not applicable </jats:sec

Phosphoproteomic and functional analyses reveal sperm-specific protein changes downstream of kappa opioid receptor in human spermatozoa

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility

O-259 Free and extracellular vesicle-associated microRNAs from endometrial fluid as non-invasive diagnostic biomarkers of implantative endometrium

Abstract Study question Is it possible to use free and extracellular vesicle-associated microRNAs from endometrial fluid as non-invasive biomarkers for implantative endometrium? Summary answer It is feasible to use free and extracellular vesicle-associated miRNAs as non-invasive tools for the detection of an implantative endometrium. What is known already MicroRNAs (miRNAs) and extracellular vesicles (EVs) from endometrial fluid (EF) have been described as mediators of embryo-endometrial crosstalk. Therefore, the miRNA analysis of EF could provide a non-invasive technique for recognizing an implantative endometrium and consequently improve implantation rates. Study design, size, duration A cohort of 162 women who assisted the human reproduction unit from January 2018 to February 2021. Of them, 72 participated in the setup and samples were collected before starting any fertility treatment in natural cycles. For the discovery of the predicted models (n = 30) and validation (n = 60), the EF was obtained from women undergoing frozen embryo transfer on day 5 and the sample was collected immediately before embryo transfer. Participants/materials, setting, methods We compared five different methodologies, two of which consisted of direct RNA extraction while the other three had EV enrichment prior to RNA extraction. A smallRNA-seq was performed to determine the most efficient method and to find a predictive model to differentiate between implantation and non-implantation endometrium. The models were confirmed by qPCR in two set of samples, discovery and validation cohorts with a different implantation outcome. Main results and the role of chance Our results showed that EV-enrichment protocols permit detection of a higher number of miRNAs. In addition, we obtained two predictive models based on three miRNAs that allow us to differentiate between an implantative and non-implantative endometrium. Results for model 1 in discovery cohort were: AUC=0.93; p-value = 0.003 and in validation cohort were: AUC= 0.69; p-value = 0.019. Results for model 2 in discovery cohort were: AUC=0.92; p-value = 0.0002 and in validation cohort were: AUC= 0.78; p-value= 0.0002. Limitations, reasons for caution One limitation to consider is the inherent variability of both the women involved in the trial and the embryos transferred. In our study, we have previously selected embryos based on morphology but we lacked genetic and molecular studies, a good complement that will certainly improve our test’s accuracy. Wider implications of the findings This study introduces new protocols to analyze miRNAs from very small volumes of EF, which could be implemented in clinical practice for the assessment of the endometrial status using miRNA-based non-invasive tools. Our results suggest that with model-2 it is possible to identify a non-implantative endometrium with 0.6-sensitivity and 0.93-specificity. Trial registration number not applicable </jats:sec