Confocal Raman spectroscopy and multivariate data analysis for evaluation of spermatozoa with normal and abnormal morphology. A feasibility study R.V. Nazarenko a, * , A.V. Irzhak b , A.L. Pomerantsev c, d , O. Ye. Rodionova c, d a EKO Infertility Treatment Centre, Moscow, 115191, Russia b Institute of Microelectronics Technology and High Purity Materials RAS, Chernogolovka, 142432, Russia c Semenov Institute of Chemical Physics RAS, Moscow, 119991, Russia d Institute of Natural and Technical Systems RAS, Sochi, 354024, Russia ARTICLE INFO Keywords: Confocal Raman spectroscopy Sperm morphology DNA fragmentation Data driven soft independent modeling of class analogies ABSTRACT This paper investigates a feasibility of using confocal Raman spectroscopy (CRS) and multivariate analysis for classification of sperm cells. The spectral based classification is compared with the morphological analysis, which is the main criterion for sperm selection in intracytoplasmic sperm injection procedure. The spectral analysis is conducted using the data driven soft independent modeling of class analogies method. The supervised classifi- cation reveals numerous outliers that pass from the 'normal' class to the 'abnormal' class, and vice versa. The ultimate result shows that the initial morphological discrimination overlaps with the spectral classification only partly. It is shown that CRS provides additional information regarding the nuclear DNA stability and helps to reveal spermatozoa with fragmented and defective DNA. This can be a promising direction for future evaluation of spectra from live, unfixed cells. 1. Introduction Over the past 25 years, Raman spectroscopy has proven to be an effective and reliable method for characterization of the intermolecular bonds [1]. This method has perspectives in the biological and medical applications. The confocal Raman spectroscopy (CRS) does not need any special conditions or sample preparation to acquire spectra. It can be used for the structural and compositional analysis of a sample before application of some destructive analytical methods [2]. The amount of the CRS applications for the investigation of tissues [3], cells [4], sub- cellular organelles and intercellular metabolic processes grows subse- quently [5]. A particular interest deserves the CRS usage in the areas of oncology [6,7], cardiology [8], and reproductive medicine [9]. Despite the fact that the results of these studies are still far from the clinical practice implementation, the available data allow us to conclude that this method has a great potential. In biology and reproductive medicine the researchers are interested in using CRS for evaluating quality of gametes. Confocal microscopes allow Raman spectroscopy to be performed with very high lateral spatial resolution and minimal depth of field (i.e., below 1 μm), thereby permitting the identification of molecules in organelles [10]. The CRS approach is especially valuable in the analysis and selec- tion of sperm for ICSI (intracytoplasmic sperm injection) procedure, because spermatozoon can be explored without harming. The studies of sperm using CRS are few so far. The sperm is a specialized, highly differentiated cell bearing the haploid number of chromosomes. The sperm main function is to deliver genetic material into the oocyte. To fulfill this task the sperm undergoes a number of significant structural and functional changes ensuring tighter condensa- tion of nuclear DNA. However, those changes have their negative fea- tures. With synthetic and metabolic processes coming to a halt in the cell, DNA is not transcribed and ready mRNA transcripts are absent due to the extremely small amount of cytoplasm. Thus, the mature sperm cell with 90–95% of its histones replaced with protamines is unable to repair the DNA damage. The DNA condensation provides a sufficiently reliable degree of protection, but if the process of histone replacement with protamines is not completed, the vulnerable DNA sites are exposed to damaging agents. Oxidative stress can lead to the single- and double- stranded DNA breaks, and the lack of DNA repair mechanisms in the sperm prevents DNA restoration. This sperm can have good morpho- logical features and motility characteristics; nevertheless, it gives rise to an embryo of a poor quality and low implantation potential. The corre- sponding embryos often stop in development [11]. Sperm DNA damage can be partially restored by the oocyte, but once the damage reaches a critical mass the oocyte DNA repair mechanism is insufficient. This fact * Corresponding author. EKO Infertility Treatment Centre, Moscow, 115191, Holodilny per.2, Bld.2, Russia. E-mail address: zdan@list.ru (R.V. Nazarenko). Contents lists available at ScienceDirect Chemometrics and Intelligent Laboratory Systems journal homepage: www.elsevier.com/locate/chemometrics https://doi.org/10.1016/j.chemolab.2018.10.002 Received 18 July 2018; Received in revised form 3 October 2018; Accepted 5 October 2018 Available online 6 October 2018 0169-7439/© 2018 Published by Elsevier B.V. Chemometrics and Intelligent Laboratory Systems 182 (2018) 172–179 This article is protected by the copyright law. You may copy and distribute this article for your personal use only. Other uses are only allowed with written permission by the copyright holder.