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

Abstract

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.

Prediction of DNA Integrity from Morphological Parameters Using a Single‐Sperm DNA Fragmentation Index Assay

“…This finding is consistent with an earlier study which showed the effect of head shape on fertilization rate . However, there is no clear conclusion that sperm head shape is significantly correlated with DNA integrity yet . In addition, our work revealed the significance and weight of other morphological parameters, such as acrosome size and acrosomal vacuole size, in predicting DNA integrity.…”

Simultaneous Morphology, Motility and Fragmentation Analysis of Live Individual Sperm Cells for Male Fertility Evaluation

“…We used a deep neural network to grade each live sperm cell according to its DNA fragmentation level. In contrast to Barnea et al [31] that shows only statistical difference for specific parameters in large populations of sperm cells measured by interferometry, with population overlaps, here we provide an individual-cell DNA measurement without staining.…”

“…Next, the solution was placed on the cover slip and stained with acridine orange (0.19 mg ml -1 ) for several hours before imaging. [31] State of the Art Fertility Examination: For each donor, 10 µL of the sample were used for stainbased morphological evaluation, 10 µL for concentration and motility evaluation, 10 µL for stain-based DNA fragmentation evaluation, and the rest was used for stain-free interferometric assays. All state-of-the-art examinations were carried out according to the WHO2010 guidelines.…”

“…For training, we used the database described in our previous work, which contains pairs of OPD maps and the same cell labeled in acridine orange and imaged under a confocal fluorescence 23 microscope (Zeiss LSM 510-META). [31] Each fluorescent cell was evaluated in a random order by the experimentalist in a color scale of 1 (red) to 5 (green). This number was used as…”

Human live spermatozoa morphology assessment using digital holographic microscopy

“…Many different variations of phase microscopes have been created 6 . In parallel, inferometric phase microscopy (IPM) with optical imaging based on digital holographic microscopy has been introduced as a mean to investigate human spermatozoa 12 , 15 . The first attempt to simultaneously assess human spermatozoa using morphology, motility and quantitative stain-free inferometric imaging analysed with a deep-learning algorithm has already been presented 16 .…”