Final Report Summary - SARM (Endometrial and embryonic genomics, searching for biomarkers in assisted reproduction (short title: Search for Assisted Reproduction Markers)) The SARM consortium (“Search for Assisted Reproduction Markers”, www.sarmproject.eu) is a partnership between five (5) European industry and academia institutions collaborating in research and technology development in reproductive medicine.The SARM consortium was funded by the EU under the Industry-Academia Partnerships and Pathways (IAPP) Marie Curie Programme (Grant: 324509, project full title: “Endometrial and Embryonic Genomics, Searching for Biomarkers in Assisted Reproduction”).The SARM consortium consists of 3 academia (Karolinska Institutet – KI, Sweden; Katholieke Universiteit Leuven – LEUVEN, Belgium; and University of Tartu – UT, Estonia) and 2 industry (Igenomix S.L. Spain and Competence Centre on Health Technologies Ltd. – CCHT, Estonia) partners. The members of the consortium join excellent and unique expertise in diverse fields, and form a strong, stimulating and coherent research environment ensuring the pooling of complementary scientific skills in reproductive medicine. Assisted reproduction is one of the fastest growing fields in reproductive medicine, which helps to overcome infertility. Infertility is the widespread medical problem seen in up to 20% of reproductive-aged couples. Its high prevalence has led to an explosion in use of assisted reproductive technologies (ART). IVF (in vitro fertilization) is the most commonly used ART technique, where oocytes are fertilized and embryos cultured in vitro and then transferred to uterus. According to European statistics, almost half a million IVF cycles are performed annually, resulting in the birth of 100,000 newborns as a 5% share from all babies. Although IVF has witnessed a rapid surge of novel laboratory and clinical technologies, the overall pregnancy rate after IVF remains only 30% per single cycle, leading to inevitable disappointment of these patients. The specific features of human reproduction are the high prevalence of diverse chromosomal pathologies in early embryos and significant dysregulation in gene expression in embryo and the endometrial, the uterine lining tissue, both being the risk factors for implantation failure and decreased pregnancy rate following IVF. The primary research objective of the SARM project was to unravel the molecular nature of human preimplantation embryo development and endometrial maturation. This ambitious goal was achieved by exploiting highly sophisticated single-cell genomics technologies, such as fine-resolution mapping of DNA copy-number changes by using single nucleotide polymorphism (SNP)-array platform and DNA-sequencing, and characterizing single-cell transcriptional status by RNA-sequencing. Our secondary intention was to combine the embryo and endometrial gene expression profiles to update molecular model for human embryo implantation. Indeed, in SARM, prominent technology and clinical innovations were made, which enabled to gain more detailed understanding of the molecular mechanisms during endometrial maturation and early embryo development and implantation, giving new hope to clinicians and their patients for improved IVF efficacy. Moreover, the SARM consortium contributed significantly to potential solutions improving safety for patients in need of IVF. Human conception is hampered by low birth rate and infertility. Early pregnancy loss is often characterized by embryonic chromosomal abnormalities. The high prevalence of chromosomal aberrations in human early embryos is a prominent feature of human reproduction. The SARM partner LEUVEN has been the pioneer in studies emphasising the alarming fact that the vast majority of IVF embryos contain chromosomal aberrations. Indeed, chromosomal instability (CIN) is a common phenomenon at cleavage-stage embryogenesis that results in an unexpectedly high frequency of chromosomal mosaicism in IVF embryos and likely explains the low success rate of IVF. Despite the high prevalence of human IVF embryos with aberrant chromosomal configurations, the preservation of CIN during prenatal development and its clinical implications are poorly understood. Furthermore, it remains unclear whether the high rate of CIN is exclusive to IVF embryos, likely due to in vitro manipulations and suboptimal embryo culture conditions, because it is impossible to investigate naturally conceived human embryos in vivo. In order to evaluate the incidence and nature of chromosomal imbalances, a novel methodology, referred to as ‘Haplarithmisis’, was developed in SARM for haplotyping and copy-number profiling of single embryonal cells. Using ‘Haplarithmisis’ we demonstrated that CIN is not preserved in later stages of prenatal development, and that de novo genomic alterations occur at similar rates in IVF and naturally conceived neonates. Thus, our findings confirm that the CIN vanishes during IVF pregnancy, and the IVF procedure has no detrimental effect on genomic rearrangements in children born, which helps to remove a major health concern for IVF neonates. The concept of ‘Haplarithmisis’ was also used in creating a new bioinformatic pipeline siChild to identify genetic defects in IVF embryos within preimplantation genetic diagnosis (PGD), the technology employed to avoid the birth of children with genetic diseases. In spite of the dauntingly high prevalence of CIN in human embryos, the reasons leading to this phenomenon have remained unclear. Due to the ethical and legal constraints associated with human embryo research, other model systems are eagerly looked for and are welcomed. Using ‘Haplarithmisis’, we demonstrated that bovine early embryonic development mimics human embryos and hence, is a good model to study embryogenesis. We confirmed a high incidence of chromosomal aberrations, but also discovered a novel cell division pattern leading to cells that carry only paternal or maternal genome, thus causing chimaerism and mixoploidy. To compare in vitro versus in vivo chromosome instability directly, we used bovine cleavage-stage embryos, by applying a genome-wide single-cell analysis method that enabled haplotyping and copy-number profiling on all individual bovine embryo blastomeres. We demonstrated that in vitro procedures exacerbate chromosome instability in cleavage-stage bovine embryos, highlighting the importance of refining assisted reproductive technologies in human. However, in the absence of human data, it is of paramount importance to propose ART only to those couples who have a clear medical indication for IVF treatment. The SARM partners have also developed a unique single-cell full transcriptome sequencing technology and implemented it in providing fundamental insight into the mechanism of human embryo development by disclosing the secrets of embryonic genome activation (EGA) that signals the transition from oocyte to embryo genome activity. These discoveries are promising for human IVF, as many embryos fail to develop because the embryo genome is not activated. However, we still miss the information on mechanisms and factors having an effect on EGA, and these studies are the topic for on-going research collaboration between the SARM partners. SARM also gave comprehensive knowledge about the processes leading to endometrial receptivity, a condition necessary for the attachment of an embryo into uterus. Still, clinically useful biomarkers for evaluating endometrial receptivity are limited, as is the understanding on the molecular and cellular processes leading to successful embryo implantation. This is mostly because in traditional studies, the genes and proteins involved in endometrial receptivity have been studied in whole-endometrial tissue biopsies, neglecting the importance of different cell populations. This fundamental barrier that precluded obtaining a more accurate view on endometrial receptivity was lifted by the seminal study by the SARM partners, where endometrial tissue was, for the first time, analysed at single-cell level. This study provided a reliable molecular tool to characterize the full cellular complexity of endometrial tissue by elucidating the roles of ‘endometrial receptivity genes’ in different cellular populations. Furthermore, we combined genome expression analyses of human embryos and endometria, and integrated these data with protein-protein interactions. These findings provided a fundamental update to the model of human embryo implantation, revealing the putative candidate genes participating in embryo-maternal dialogue.Besides outstanding contribution to the scientific understanding of human reproduction, the SARM consortium provided the possibility for more profound integration of European research at both academia and industry levels, leading to more efficient and systematic transfer of knowledge and skills between the two sectors. SARM partner Igenomix is a Spanish company with broad experience in pioneering genetic and molecular diagnostics in Europe. Igenomix is globally known for its ‘Endometrial Receptivity Array’ (ERA) test, which is successfully used in female infertility diagnostics of endometrial origin. In the SARM project, novel endometrial receptivity biomarkers were identified, and the next-generation sequencing based ERA testing was adapted. Moreover, Targeted Allele Counting by sequencing (TAC-seq) a novel proprietary molecular technology was developed by partners CCHT and KI for cost-effective and quantitative analysis of endometrial receptivity biomarkers for infertility diagnostics. Relating to the secondment and recruitment of fellows – the main deliverables of the SARM IAPP project –, within 4 years, 20 fellows were seconded and 7 fellows recruited, for 168.62 and 96.79 months, respectively (in total 27 researchers for 265.41 months). The gender balance has been towards the female, having 10 male and 17 female fellows involved.In conclusion, the SARM project covered the clinically urgent aspects in women’s reproductive medicine, including infertility and the origin of fetal/newborn diseases, while also devising novel technologies for treating infertility and implementing genetic technologies to prevent birth of children with severe diseases. In the SARM studies, we emphasised the continuum of processes in early embryo development, which are vulnerable processes due to different molecular aberrations, like chromosomal instability, may occur and lead to implantation failure, miscarriage or even a birth of a sick child.The SARM research has the largest impact on families experiencing infertility, as a substantial part of the research aimed at more reliable, cost-effective and safe tools for infertility treatment.
