Multiparametric analysis of sperm chromatin and ROS reveals the existence of subpopulations that are linked to embryo...

Sperm DNA damage is known to be linked to fertility potential, yet the structural integrity of sperm chromatin has received less attention in male factor research. The objective of this work is to develop a multiparametric test to simultaneously assess chromatin protamination, chromatin condensation, reactive oxygen species (ROS), and viability in human sperm; to explore whether sperm subpopulations based on chromatin integrity and ROS levels can be identified; and to evaluate their impact on fertilization rates and embryo development after ICSI. For this purpose, cryopreserved sperm from 43 donors involved in 47 ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A) were analyzed. The degrees of chromatin protamination (Chromomycin A3, CMA3) and condensation (dibromobimane, DBB), ROS (H₂DCFDA) levels, and viability were determined simultaneously through flow cytometry. After incubation at 37 °C for 4 h, viable sperm exhibited increased chromatin condensation and a mild rise in ROS levels (P < 0.0001). Principal component and cluster analyses were performed to identify distinct sperm subpopulations, and linear regression was used to assess associations between sperm parameters and ICSI outcomes. We identified three sperm subpopulations based on ROS levels, and chromatin protamination and condensation. The first sperm subpopulation presented low ROS and high degrees of chromatin condensation and protamination. The second showed a moderate increase in ROS and chromatin decondensation, but a normal degree of chromatin protamination. The third exhibited high ROS, and poor chromatin protamination and condensation. Interestingly, the percentage of sperm belonging to subpopulation 2 was positively associated with the rates of total blastocysts (P = 0.012) and good-quality blastocysts (P = 0.028), whereas that of sperm belonging to subpopulation 3 was linked to delayed embryo development (P = 0.016). In contrast, percentages of sperm belonging to any of these chromatin-ROS subpopulations were not associated with the proportion of aneuploid blastocysts (P > 0.05). In conclusion, our multiparametric analysis suggests that the percentage of sperm in subpopulation 2, which exhibit highly protamined but moderately decondensed chromatin and mildly increased ROS levels, is associated with improved blastocyst development. In addition, our data support the hypothesis that these sperm may retain the capacity for chromatin overcondensation. Finally, our approach underscores the relevance of sperm chromatin protamination and condensation to fertility potential and underpins further examination of these factors in the context of ICSI.