Chromatin Maturity Index (CMI) in unfixed and live spermatozoa and Aniline Blue (AB) stained as an additional evaluation parameter in idiopathic male infertility

Study question: To investigate whether idiopathic male infertility may be due to the presence of histones in motile spermatozoa using a modified AB staining protocol. Summary answer: No correlation between CMI in live motile spermatozoa, DNA Fragmentation Index (DFI) and other conventional seminal parameters were found in male infertile patients. What is known already: The AB stain discriminates between lysine-rich histones and arginine/cysteine-rich protamines. Transition from histones to protamines during spermatogenesis remodels chromatin packaging and abnormalities in the substitution of those proteins maybe interfere with seminal parameters and affect male infertility. The correlation between CMI and seminal parameters is known, but little is knowledge about live and motile spermatozoa associated to CMI because literature report only spermatozoa fixation before staining. Sperm chromatin carries half of the genomic material to offspring. Spermatozoa nuclear status is crucial for balanced transmission to future generations, and histones modifications are directly involved in epigenetic mutations. Study design, size, duration: Retrospective observational study of 77 men underwent to standard semen analysis, including the evaluation of CMI and DFI, enrolled from January to December 2020. Mean age of the men was 36.63±8.26 years old, sperm concentration 46.69±37.23 mill/mL, linear progressive motility 39.35±15.31%, normal morphology 6.42±3.40%, DFI 25.91±10.29%. 200 spermatozoa for evaluation of CMI and 300 for DFI were analyzed respectively. Participants/materials, setting, methods: Semen samples of 77 patients were collected and analyzed according to 5th edition of WHO guidelines (2010) for examination of human semen. For the evaluation of CMI we performed a new modified protocol for AB stain directly in live spermatozoa. Dilution 1:1 fresh semen and Aniline Blue colorant were mixed and placed on a slide and examined in bright field microscopy x1000 magnification. DFI was evaluated using Sperm Chromatin Dispersion (SCD) test. Main results and the role of chance: Of all spermatozoa analyzed, 82.58±29.98% were white, 17.17±17.21% were pale blue, and 28.53±21.09% were dark blue. By our modified protocol, directly in live spermatozoa, we correlated AB staining with motility and , surprisingly, all motile spermatozoa observed were not stained (white), while pale or dark blue spermatozoa resulted always immotile. For this reason, we have considered pale blue spermatozoa as AB positive, in disagreement with some authors. So, maybe, we should reconsider pale blue stained spermatozoa as abnormal. We also observed AB negative spermatozoa with morphological head, neck and tail defects, underlining the independence of these two parameters: nuclear status and morphology. We have observed no statistically significant differences between conventional semen parameters, DFI and CMI, so nuclear analysis seems to be independent parameters. The statistical analysis was performed by Matlab statistical toolbox version 2008 (MathWorks, Natick, MA, USA) for Windows at 32 bit; finally all tests with p-value (p) < 0.05 were considered significant. Attention should be paid to the evaluation of CMI not only in astenozoospermic patients, where a lower CMI is known, but also in normozoospermic infertile patients. Limitations, reasons for caution: This is a preliminary observational study on a small number of normozoospermic or mild asthenozoospermic patients. The study should be considered as a pilot study. Future studies with higher number of samples are necessary in order to confirm the results obtained. Wider implications of the findings: This is the first study that reports AB staining on unfixed live spermatozoa with a modified protocol. Our study underlines the necessity of classify pale blue spermatozoa as AB positive. Further investigations are necessary. This is a starting point for future analysis to be carried out under the project EcoFoodFertility. Trial registration number: not applicable