P–046 Application of hyperspectroscopy as a new sperm diagnostic and selection technique for ICSI based on hyperspectral signature of single spermatozoa – a proof of concept
Authors: VILLANUEVA E , Gi. Julia M , lo. Santos J M D , Hervas I , Navarro-GomezLechon A , Rivera-Egea R , Garrido N 
Published: August 2021

Abstract

Study question
Is hyperspectral imaging of individual sperm cells an appropriate candidate technique with potential to identify molecular characteristics of single spermatozoa prior to microinjection?

Summary answer
Preliminary tests have allowed us to obtain the images, set up the methodology and the correct segmentation of each sperm into its signature spectra.

What is known already
Although image techniques such as fluorescence microscopy and Raman spectroscopy have been used to identify biomarkers on sperm cells, their translation to the fertility clinic is highly complex and unfeasible. Hyperspectroscopy has not yet been introduced in the reproductive field, but it has been successfully used in other medical disciplines to differentiate cell types or pathological versus healthy tissue. It relies on the combination of information from optical images and electromagnetic spectra produced by cells at different wavelengths. Therefore, it is an interesting candidate for objective non-destructive sperm selection before ICSI according to the presence of specific biomarkers.

Study design, size, duration
Pilot, prospective, observational study in the IVIRMA Valencia fertility clinic, designed as a proof of concept. Discarded semen samples were used for the technical setup and the preliminary tests of image acquisition. Hyperspectral images were obtained of 12 spermatozoa: two sperm cells were imaged 10 times each, while the other 10 sperm were imaged one time each.

Participants/materials, setting, methods
Samples were prepared on glass-bottom ICSI plates in PVP droplets according to routine clinical practice. Images were obtained using the camera HinaLea 4200M coupled to the Olympus IX73 inverted microscope. Hypercubes were extracted using MATLAB 2016a, and the most informative wavelength was chosen. Matrox Copilot software was used to process images and select the regions of interest (ROI) equivalent to one sperm. MATLAB was used to obtain the information for the pixels within each ROI.

Main results and the role of chance
Hypercube size for each image was 608x968x299 pixels. Each hypercube obtained provided information for 299 different wavelengths, from 402.8 to 998.3 nm. Once the hypercubes were obtained from each image, the spectra at 520 nm wavelength was selected as the one at which the pixel provided the most information. The background of each uploaded image was successfully removed by creating a mask, which was then used to extract and characterize all the spectra from each pixel of each sperm image. The result of this proof of concept, though measure qualitatively, is that the information obtained from hyperspectral imaging of individual sperm is highly reproducible and successful in showing overlapping spectra resulting from 10 different images of the same specimen. Spectra resulting from images of different sperm cells showed tangible differences on their composition demonstrating unique biochemical features of each. The next step will be to quantitatively measure the reproducibility and sensitivity of the technique in a larger sample, to then study the potential of single sperm hyperspectral imaging as a sperm selection tool for ICSI.

Limitations, reasons for caution
These are preliminary results obtained from the development of an appropriate methodology to adapt the acquisition of hyperspectral images in the clinic’s IVF laboratory. Thus, the group will continue to evaluate the reproducibility, sensitivity, and informative capability of the technique before contemplating its use on samples destined to perform cycles.
Wider implications of the findings: Having developed a laboratory and image processing methodology that allows us to successfully segment each obtained image according to the hyperspectral information of every pixel of each individual sperm, we could move forward in testing the potential of these hyperspectral signatures to identify sperm markers related to successful reproductive outcomes.

Trial registration number
Not applicable