Stain‐free interferometric phase microscopy correlation with DNA fragmentation stain in human spermatozoa

Acridine orange (AO) staining is used to diagnose DNA fragmentation status in sperm cells. Interferometric phase microscopy (IPM) is an optical imaging method based on digital holographic microscopy that provides quantitative morphological and refractive index imaging of cells in vitro without the need for staining. We have imaged sperm cells using stain-free IPM in order to estimate different cellular parameters, such as acrosome dry mass and size, in addition to an embryologist evaluation according to the World Health Organization (WHO)-2010 criteria. Following this, the same sperm cells were stained by AO, imaged using a fluorescence confocal microscope and assessed by the AO-emitted color, forming five DNA fragmentation groups. These DNA fragmentation groups were correlated with the embryologist-based classification and the IPM-based morphological parameters. Our results indicate on significant differences in the IPM-based parameters between groups with different fragmentation levels. Based on the validation with AO, we conclude that stain-free IPM images analyzed digitally may assist in selecting sperm cells with intact DNA prior to intracytoplasmic sperm injection. This information may potentially increase percentage of successful pregnancies.