Early pregnancy loss (EPL) in horses, with an incidence rate of 5-10%, is a significant challenge for equine reproduction. EPL leads to higher breeding costs and reduced reproductive efficiency yielding fewer healthy foals. Chromosomal abnormalities, especially aneuploidy (loss or gain of chromosomes), are responsible for over 55% of EPL cases in Thoroughbreds. Certain chromosomes (3, 4, and 20) are frequently involved, suggesting a specific genetic or structural issue within these chromosomes that contributes to EPL. Understanding the molecular causes of aneuploidy is essential for improving breeding strategies and ensuring equine reproductive success, and will be a main focus of this proposal.  One key driver of aneuploidy may be the centromere, a chromosome region crucial for proper genetic inheritance and healthy pregnancies. Studying centromere genetics in relation to EPL could lead to diagnostic tests for predicting EPL risk, improving pregnancy outcomes, and reducing costs. Currently, the genetic factors that increase the likelihood of chromosomal errors in EPL are not well understood. Our goal is to identify structural and genetic centromere variants in equine chromosomes, particularly in Thoroughbreds, that make embryos more prone to EPL.  Centromeres are characterized by the presence of a chromosome packaging histone protein, CENP-A, often associated with repetitive DNA. Our strong preliminary data using an established bioinformatics pipeline, has shown that centromere DNA is highly diverse in its repeat composition and copy number in Thoroughbreds. We hypothesize that this incredible variation in centromeric DNA impacts chromosomal error correction during cell division, leading to a higher risk of chromosomal abnormalities. Indeed, Dr. Das' previous work revealed that diverse centromeric genetic variants significantly impacts biological functions in other mammalian embryos. This project will use computational and cell biology methods to analyze centromere genetics and function across different equine breeds to identify centromere variants enriched in specific chromosomal aneuploidies associated with an EPL case. This study is a close collaboration between Ors. Das and de Mestre. Dr. Das brings expertise in chromosome biology, embryology, and genetics of repetitive genomic regions, while Dr de Mestre brings significant expertise pioneering the methods to study and analyze conceptus tissues following EPL, a unique bank of clinical material collated in her laboratory over 10 years, and experience in molecular reproductive biology and genetics in EPL.  This research is a critical step towards understanding the genetic basis of EPL and developing biomarkers to guide equine breeding decisions. The findings could be applied across various equine breeds to improve pregnancy management. Additionally, we will create a detailed atlas of all centromere variants, including ones enriched in aneuploid EPL cases, providing a valuable resource for future studies on the genetic elements underlying EPL.