Early pregnancy loss (EPL) remains one of the greatest challenges in equine reproductive health as it is very common and in the majority of cases the underlying cause is not identified. The overarching goal of this project is to identify inherited and congenital causes of equine EPL, here defined as loss in of a pregnancy within the first two months of gestation, with losses after clinical detection (day 11-15) the focus of this study. We recently identified the structural genetic variant, aneuploidy, as the most common cause of EPL described in the mare to date. The role of other structural genetic variants involving smaller segments of the chromosome of 1kb to MB in length (Copy Number Variants or CNVs), remains unknown, but have similarly been shown to result in fetal lethality in humans. In our preliminary studies, we have identified several strong candidate lethal CNVs in clinical cases of EPL submitted to our laboratory that result in deletion of a region possessing a key developmental gene and found exclusively in EPL conceptuses but not viable individuals. We hypothesise microdeletions (CNVs involving a deleted copy of the genome) present in embryonic and early fetal stage and encompassing key developmental genes can impact early development and lead to pregnancy loss in the embryonic and early fetal period. We predict these microdeletions will be lethal if they involve loss of both alleles or loss of a single allele that overlaps a haploinsufficient gene. We will address this hypothesis using tissues isolated from clinical cases of EPL submitted to our laboratory in 2023, with additional submissions committed for 2024/2025. In aim 1, we will identify novel CNVs unique to EPLs that encompass key developmental genes and impact gene dosage. We will achieve this aim through a combination of PacBio Revio long read sequencing to map the co-ordinates of three candidate CNVs identified in preliminary studies, digital droplet PCR to demonstrate the penetrance of the CNVs in EPLs and absence in all viable controls, and length extension ChRO-Seq to determine if the CNVs encompass transcriptional active genes or regulatory elements that are subject to gene dosage. MicroCT will be applied to screen the EPL fetuses for developmental abnormalities linked to the function of the impacted gene. In aim 2, we will determine if the CNVs are inherited through the germline or introduced in cell division during early embryonic development using digital droplet PCR and screening for the CNV across tissues within a EPL and controls as well as determining if the sire and dam of the EPLs are carriers of the CNVs. Dr de Mestre brings significant expertise pioneering the methods to study and analyse conceptus tissues following EPL, a unique bank of clinical material collated in her laboratory over 10 years, expertise in molecular reproductive biology and genetics and strong preliminary data supporting the involvement of CNVs in EPL. She has teamed with Dr Danko and his laboratory who have led the field in the development of in unique chromatin run on assays that assess regulatory elements and transcription. We expect the outcomes to form the basis of a novel diagnostic test to identify CNVs as an underlying cause of early pregnancy loss that is currently unknown in approximately 60% of clinical cases.