While an increasing number of infertile patients seek therapies using assisted reproductive technologies (ART), the success rates for these techniques have not dramatically increased in the past decade [1]. In cases of male factor infertility treated with intracytoplasmic sperm injection (ICSI), the choice of which sperm to micro-inject may influence the clinical outcome [2]. Several studies have suggested that microscopic selection of sperm, based upon morphology, could potentially increase ICSI success ([3, 4] however, this selection is subjectively determined by an embryologist. Analysis of sperm from fertile and infertile men have revealed that a wide variety of parameters measured in human sperm vary substantially, with some correlating with fertility via natural conception or ART. However, to date functional data linking objectively identified sperm sub-populations to successful therapeutic outcomes remain sparse. Work from my laboratory has pioneered the usage of fluorescence activated cell sorting (FACS) to isolate sperm from patients based on impermeability to a nucleic acid dye (YO-PRO-1). Sperm selected in this way exhibit decreased levels of DNA fragmentation ([5], and have been used in a randomized clinical trial (RCT) to successfully generate healthy full-term offspring (De Geyter et al. submitted). Despite these positive preliminary results, further improvements in ART success rates are needed.Here we propose a series of detailed analyses on sperm of age-matched fertile and infertile men to determine the co-occurrence of known markers of ART failure on individual sperm. We will predominantly focus on factors involved in chromatin compaction and epigenetic status of sperm genome, which is considered to regulate gene expression in conceived embryos. We will therefore compare sperm of two groups of infertile men who differ in their ability to support pre-implantation development after fertilization. The first cohort corresponds to men of couples whom were previously treated with ART, but in whom fewer than the expected number of embryos developed to the blastocyst stage. The other half of the infertile men will be selected from a cohort of couples previously treated with ART, in whom more than half of the fertilized oocytes developed to the blastocyst stage. By using image quantitation pipelines, we will expand the reproducibility and sensitivity of such assays, determining whether combinations of measurements provide improved prognostic outcomes. We will also investigate a series of novel nucleic acid dyes to examine unique features of sperm chromatin. Following our identification of subpopulations of sperm, we will use FACS to isolate these populations for further characterization. We will perform extensive molecular characterizations of prominent epigenetic markers in the sperm genome. We will determine the genome wide distributions of histone proteins, their modifications and of DNA methylation as well as global RNA expression signatures in sub-populations of human sperm. Through such comparative genome-wide studies, we aim to identify the molecular foundations of sperm variation, potentially providing molecular explanations for the majorly different efficiencies in driving embryonic development among men with seemingly normal semen.