Human live spermatozoa morphology assessment using digital holographic microscopy

Abstract

Digital holographic microscopy (DHM) was applied for the morphological assessment of live intact spermatozoa from fertile and infertile men directly after semen liquefaction. This method allowed us to study the sperm population directly from the sample droplet and not only from the focal plane of the microscope as in classical optical microscopy. The newly implemented 3-dimensional sperm morphological parameters (head height, acrosome/nucleus height, head/midpiece height) were included in morphological assessment of semen samples from fertile and infertile individuals. The values of the 3D parameters were less variable in fertile men than for infertile ones. DHM was also used to compare the morphological profiles of spermatozoa after applying the "swim-up" and gradient centrifugation techniques. During selection, the most statistically significant differences were observed after separation with a Percoll gradient of 90% and a 60-min "swim-up" procedure versus 'native' unfractionated samples. This shows that the developed methodology can be efficiently used for the selection of morphologically sound spermatozoa. The motility type for each spermatozoon was also assessed. The results indicate that the extension of the number of morphological parameters with new 3D parameters and the simultaneous assessment of sperm motility may be valuable addition to sperm examination.

Figure 1

Comparison of spermatozoa 2D and 3D morphological parameters in fertile and infertile men by using DHM (digital holographic microscopy). Panel I (3-D parameters), Panel II (2-D parameters).

Figure 2

Data range of novel spermatozoa parameters measured by DHM for fertile and infertile men. (A) Range of values for hh in fertile men (median-1.90 µm) narrowed down in a statistically significant manner compared to the infertile group (median-2.61 µm) (P < 0.05). (B) Range of values for amh (acrosome/nucleus height) narrowed down in a statistically significant manner for the group of fertile men (median- 1.69 µm) vs. infertile individuals (median- 2.34 µm) (P < 0.002) (C) The median of hmh for fertile men was 1.30 µm while that for infertile individuals was – 1.47 µm.

Figure 3

Holographic imaging of spermatozoa obtained via DHM. (A) Two phase images based on one hologram with sharpness at different sample depths. (B) Two computerized 3D reconstructions based on one hologram with sharpness at different sample depths. (C) 3D reconstructions of a single spermatozoon.